A Mighty Demon Slayer Grooms Some Ponies is about ponies deciding to make First Contact with humanity by participating in a horse show, and for that they need to be well-groomed. Grooming is treated with varying levels of mock-seriousness throughout the story, including one character stating that "the future of two worlds depends on you having a well-brushed coat".
In The Universiad, a Spacebattler story the Forum once got into a civil war over paper. How serious? It lasted 15 years and the Originals still pay remembrance by having annual 15-day long live-fire "reenactments".
The voice synthesizing software Vocaloid and its characters. The only thing canon about the Vocaloid universe by Word Of God is the character names, designs, and voice banks, but the Fanon is quite firmly rooted, to the point where one thing that was officially canon got retracted because of fan outcry. The software and the fandom it spawned has inspired countless songs, and those songs have inspired a stage play, a musical (inspired by only a single song, mind you), and an anime. The flagship character, Miku Hatsune, is a bona fide Virtual Celebrity, complete with an album reaching # 2 on Japanese charts. She also gave a live concert (as a hologram, backed by live human musicians), and her version of the software has also been installed in a Japanese gynoid.
In Doonesbury, the frenzy over Beanie Babies (see the Real Life section) was mocked in a storyline that saw Uncle Duke steal Alex Doonesbury's collection and hold them for ransom. The whole thing is treated as seriously as an actual kidnapping.
In the Final Battle of Dino Attack RPG, when a large number of Dino Attack agents used the comm chatter to crack jokes or make silly references, one Dino Attack agent attempted to Shoo Out the Clowns by telling them to stop making jokes and references and start concentrating on the battle, using the comm chatter only for coordinating attacks and strategies. This example is largely Played for Laughs, since this one Dino Attack agent is apparently the only one trying to use the comm chatter for its intended purpose in the battle, yet everyone completely ignores him (or, in one agent's case, openly mocks him with a quote from Monty Python's Flying Circus) and continues using the comm chatter for less-than-serious commentary.
Unfortunately, this was very much the case Out of Character in Campus Life for a good long while. Pretty much whenever someone does something that even slightly annoys another role-player or breaks the narrative flow too much, you can pretty much guarantee a massive fight's about to break out in RP Discussion. At one point it got so bad a user ended up getting banned for three days and the current Admin at the time experianced a massive Creator Breakdown that resulted in the RP getting ported over to another Forum. Though things have calmed down quite a bit since then, it still tends to pop up every once in a while.
In the storylines of many ballets, Dancing Is Serious Business. The hero of Swan Lake dooms his beloved to spend eternity as a swan because he mistakenly dances with the wrong woman at a ball. The titular heroine of Giselle dances herself to death, and later spares the man she loves from the same fate by offering to dance in his place to appease an evil ghost queen who is forcing him to dance again and again. In The Sleeping Beauty, Aurora pricks her finger not from spinning, but from dancing with the spindle despite her mother's warnings that doing so would be dangerous.
The Arbiter in the musical Chess not only takes his job of refereeing a chess championship incredibly seriously, he also seems to think it makes him a badass. "I'm on the case, can't be fooled/ any objection is overruled/ I'm the Arbiter, I know the score/ from square one/ I'll be watching all sixty-four..."
It's really the music itself that turns the whole thing into Serious Business. Bangkok, anyone?
Red vs. Blue; the continuing story of a battle fought over "the galaxy's most important box canyon with a base at each end." (There was more here, but it seems to have been lost. Anyone?)
The others still appear to be referred to as privates when not being addressed by name, so either it was a throwaway line that was just Wash being insulting, or the writers themselves forgot Wash said it.
The red and blue teams are simulation Troopers. They are a part of the Army, just a part where the soldiers are considered expandable. Its also hinted that the simulation war was a testing ground for Project Freelancer exclusive, which they used to test stuff, train their Agents and recruit new Soldiers(as seen with Donut).And to hide the Alpha.Sarge still qualifies as an example, since no one else of the Blood Gulch Crew takes the war serious.
In one PSA, Grif states that "some games are serious business." Apparently, he once "played Donkey Kong so well that he cured kidneyism. It was the best day ever. The End."
DigitalPh33r regularly parodies the concept in his Halo movies with unnamed characters brutalizing things in game and/or shouting to the heavens "THIS IS SUCH A BIG DEAL!!!"
The classic Flash film Craziest is about someone who considers Scrabble a religion.
TV Tropes is serious business. When you think about it, this site's entire premise takes fiction as Serious Business. Let us please leave it at that.
Taking irony to new levels of meta, this entry has become Serious Business.
It's slightly terrifying that there are five pages of people arguing about a basketball player's neutrality and favorite things.
Once in a while, certain changes to Wikipedia or references prove how MUCH of a Serious Business Wikipedia is. A recent example - look up "Malamanteau" on Wikipedia and go to its Talk page. It's incredible how a single joke can provoke a reaction of such scope.
Also, certain data being on the article is serious business to some people, as the Lamest edit wars page shows.
Certain celebrities and politicians have regarded the wiki as significant enough to embellish their own entries or create and maintain their own pages in defiance of the editing policy. To their credit, the administrators tend to snuff out such shenanigans quickly enough.
If the topic is of the serious business type (sciences and such), you can use it as a source hub.
My Life Is Average has a very dedicated group of people that hate on any story that isn't average or has words like "it made my day" or "Best. X. Ever."
deviantArt is taken very seriously at times. Granted some of it is over important stuff like art thievery, but then things just go a little nutty over other topics, such as whether one should comment on a piece before faving it or if the favorite is a comment by itself.
Heaven help you if submit a piece in the wrong category.
The IMDB Top 250. That is all. Sometimes it even gets rigged to help certain movies. Voters have admitted to giving certain movies a "1" or a "10" just so they can bring the average score closer to their desired number.
Facebook. Your parents and grandparents will know everything you do and will get offended at you. Employers will check your facebook first before anything else. And heaven help you if you ever defriend someone.
Confusing "your" for "you're" on a forum. Grammar nazis will pop out of nowhere to give you a tongue lashing hazardous to your health.
Any website with user-generated content and a point system to reward those who contribute the most has the potential to turn a fairly meaningless number that is only occationally noticed by a handful of people into Serious Business.
ScrewAttack's Mario Party After Dark will usually (very quickly) escalate into all-out war between the guys. They even have a saying regarding it: "Check your friendships at the door."
Journal Roleplay games can get this way, too. To the point where there's a Dreamwidth community, the anonymous-based "wankgate", dedicated to pointing out any and all flaws in certain players and games... then attempting to either get them to clean up their act in the most vile way or drive them off of the face of the Internet in eternal shame.