The sequence on Earth-13122, the Lego universe, was animated by 14-year-old Preston Mutanga, who was hired after Phil Lord and Christopher Miller were impressed with his Twitter video recreating the entire trailer with Lego.

Phil Lord and Christopher Miller revealed that they told Sony the sequel would be the same size as Spider-Man: Into the Spider-Verse (2018), but it ended up having the largest crew of any animated movie ever with around 1,000 people working on it. They added that it has 240 characters and takes place in six universes.

Spencer Wan, one of the animators for the film, used spam emails he received as background textures for Hobie Brown's animation throughout the film.

At a runtime of 2 hours and 20 minutes (140 minutes), this was the longest American animated film at the time of its release, surpassing the previous record holder, Consuming Spirits (2012), by 4 minutes. The record was surpassed the following year by Hello My Beautiful Creatures (2024), at 2 hours and 22 minutes (142 minutes), though that film had a lower frame rate. Across the Spider-Verse is the third animated film produced by a major American studio to run over 2 hours, after Fantasia (1940) and The Lord of the Rings (1978).

When Miles is rushing to meeting with the guidance counselor, he passes by his friend Ganke who can be seen hanging out in Miles' and Ganke's dorm room playing Marvel's Spider-Man 2 on his PlayStation 5. The game's version of Spider-Man, played by Yuri Lowenthal, also appears as one of the 240 Spider-Man variants seen throughout the film.

Metro Boomin: Music producer and soundtrack contributor appears as "Metro Spider-Man," who tells Miles that "there's nowhere to run."