In the days after the attack on Pearl Harbor. Lt. Col Jimmy Doolittle (Tracy) assembles a group of volunteers to man B-25 bombers for a mysterious mission. Among those pilots is Lt. Ted Lawson (Johnson), who just got married a few months ago and whose wife Ellen has just told him she's pregnant. Johnson and his crew—co-pilot Dean Davenport, bombardier Robert Clever, navigator Charles McClure, and tailgunner Cpl. Robert Thatcher (Walker)—start practicing unusually short 500-foot B-25 takeoffs. Lawson's crew and all the other crews get a clue when a naval officer shows up to coordinate their drills, but not until their planes are aboard the carrier USS Hornet and the men are out to sea are they told their mission. They will be taking off from the Hornet and conducting the first Army Air Force bombing raid of Japan. And because they are taking off from so far out, it's a one-way trip. The men will have to land somewhere in China (if they make it that far), evade capture by Japanese forces in China, and make it to safety in the Nationalist Chinese capital of Chongqing.
The Doolittle Raid inspired no fewer than four films, including Cary Grant vehicle Destination Tokyo, but the other films were heavily fictionalized, while Thirty Seconds Over Tokyo was made to be an accurate account of the real history of the raid. Robert Mitchum had one of his first big parts in this film, as another bomber pilot.
- An Arm and a Leg: Lawson badly injures his leg in the crash landing. Eventually it has to be amputated.
- And Starring: Spencer Tracy only has a few scenes—Van Johnson is the star as Lawson—but in the end credits after all the other names, he gets a large-type "And Spencer Tracy as Lieutenant Colonel James H. Doolittle" credit.
- Based on a True Story: Says so in the opening title, and in fact is a pretty accurate history of the Doolittle Raid and the experiences of Lt. Lawson and his crew.
- Buzzing the Deck: Sort of. They aren't trying to scare anybody, but when flying through San Francisco Lawson flies his plane under the Bay Bridge, on a dare.
- Cigarette of Anxiety: After Doolittle tells the men that they will be going on a one-way bombing run over Japan, a visibly shocked pilot stands up and asks Doolittle if it's OK to smoke during briefings.
- Country Mouse: Thatcher, the rube from Billings, Montana, takes a lot of ribbing from his more worldly fellow airmen.
- Earn Your Happy Ending: After a premature takeoff, a bombing raid, a crash landing on the Chinese coast, a desperate flight from the Japanese, and the amputation of his leg, Lawson makes it home, as do his men. (In Real Life, 69 out of 80 men survived the raid and made it back home. Three were killed in action, three were captured and executed by the Japanese, one died in Japanese captivity, and four more survived in Japanese POW camps until the end of the war.)
- Fighter-Launching Sequence: The pilots take off from an aircraft carrier in B-25s, something that had never been done before. It led to this exchange between a pilot and co-pilot.Lawson: Raise the flapsDavenport: (Looks) They weren't down.Lawson: Great. I wonder what else we forgot.
- Fish out of Water: The airmen are out of their element on a Navy carrier. Some of them get lost trying to find their way below decks.
- Flashback: A dazed Lawson has a flashback to his wedding, while lying on a Chinese beach in great pain following a crash landing. Later, as his leg is getting cut off, he has a flashback to the time he called Ellen the previous Christmas.
- Hide Your Pregnancy: In-Universe, as Ellen, nervous about seeing Tom again now that she's fat with pregnancy, asks her mother if holding a coat in her arms is sufficient to hide the baby bump. This is made more poignant by the fact that Ellen is aware that Tom lost a leg.
- It Has Been an Honor: Not right before the raid. But after explaining the nature of the mission, Doolittle ends his briefing by saying "You're a great bunch of guys. I'm proud to have been associated with you."
- I Will Only Slow You Down: Lawson, laid up with an injured leg that eventually has to be amputated, tells the other men to get out before they're captured by the Japanese. They ignore him, and eventually they all make it to safety.
- Nose Art: Lawson's plane ("ship"), the Ruptured Duck, has a drawing of...Donald Duck with crutches◊.
- Shown Their Work: The practice scenes were shot on the real airfields where the Doolittle Raiders trained, using real B-25 bombers. An aircraft carrier set big enough to hold four real planes was built. Most impressively, footage of the real Doolittle planes taking off from the Hornet was used in the movie.
- Single Tear: Seen on the face of a single old Chinese lady as the locals help the Americans out of their village.
- Someone to Remember Him By: Discussed Trope, as Ellen muses over how if Ted is killed, she'll still have a little piece of him, in their baby.
- The Squad: A five-man B-25 bomber crew. Lawson's the leader, the co-pilot likes to play poker, the bombardier is a home movie enthusiast, and Thatcher is a hick who proves unexpectedly tough under pressure.
- That's an Order!: Doolittle says this when making it clear that the volunteers for what became the Doolittle Raid are not to discuss where they're going with anyone.
- What You Are in the Dark: Played for a gag. Lawson is getting a bunch of cigarettes from the ship's store when the battlestations alarm sounds. He runs out, doubles back, picks up the cigarettes, picks up the money that he'd dropped on the counter, then puts the money back down.