Ebony White, The Spirit's sidekick from the comics, was omitted, largely because he was typically portrayed as a 1930's racial stereotype. Frank Miller disliked the idea of The Spirit having a sidekick, so the character was completely removed, rather than updated.
There are several references to other comic book characters throughout the film: Sand Saref mentions Robin, Morgenstern mentions the Electra complex (based on the daughter of Agamemnon, the basis for the Marvel character Elektra), and The Spirit mentions Thor's hammer.
Stana Katic's character Morgenstern gets told twice: "You'll make detective in no time." and in the television series Castle (2009), her character Kate Beckett is the youngest woman to ever make detective in the NYPD.
In an interrogation scene, the Spirit asks the Octopus "What's in the vase, Thor's hammer?" Samuel L. Jackson (The Octopus) and Scarlett Johansson (Silken Floss) later appeared in Marvel's The Avengers (2012), (as Nick Fury and Black Widow, respectively), which also features Thor (played by Chris Hemsworth) as a member of the team.