Batman and Harley Quinn is far more comedy-driven compared to most of the more recent DCAUO-films, but there are some sadder moments in the movie:
- As Harley explains to Nightwing while she's holding him hostage, even though she's turned over a new leaf (for the most part at least), she's unable to find a decent job because of her criminal record—pretty much the only jobs she can find (outside of her current occupation) is basically just being a porn star or a stripper. Even worse, this is very much Truth in Television, as a lot of places won't hire anyone with a criminal record, which often results in them returning to their life of crime out of desperation. Even worse when you realize that she did return to crime, as this film takes place before Joker's death, as seen in Batman Beyond: Return of the Joker.
- When Batman and Nightwing break into Jason Woodrue and Poison Ivy's hideout, Ivy is hilariously shocked to see that Harley has joined up with the Dynamic Duo. Then comes the line "Oh, Harl..."; Paget Brewster clearly worked really hard to put a genuine depth of betrayal into those two words.
- The look on Ivy's face when Jason Woodrue forces her to leave Harley for dead in the fire. Then that is followed up by Harley's sheer stare of abandonment.
- Dr. Harold Goldbum's death is definitely the saddest scene in the entire film.
- Harley breaking down in tears when Batman demands she stay behind when he and Nightwing are off to Louisiana. Played for Laughs, yes, but still heartbreaking when she mentioned all the pets and plants she had over the years that died and that she'd probably forget to water herself if she became a plant-hybrid. Nightwing has to talk Batman into letting her tag along.
- The part in the climax where Harley uses Puppy-Dog Eyes and Cry Cute on Ivy is sad in itself when you think about the Fridge Brilliance behind it. Ivy's seen Harley cry and mope before, so you might wonder why it can bother her now. Then it hits you; Harley is displaying to Ivy the very last thing she wants to see: the pain and trauma Harley endured from the Joker, now, at Pamela's hands.