Here's #38! Click here for the master list!
Detective Comics #38
While there may be a real challenger that I am overlooking, I am pretty sure that this is one of the clearer decisions on the list, as the first appearance of Robin in Detective Comics #38 is not only extremely important, comic history-wise, but the cover of the comic is also a total classic.
There had been kid sidekicks before in fiction, of course, but this was the first kid sidekick to a superhero, and soon, basically EVERY hero had to have one (with Superman and Wonder Woman being the notable exceptions - and I guess Namor didn't have one, either).
Not only is it an important issue, it is also a good one - Bill Finger presents a strong debut for Robin (after Jerry Robinson gave him a strong foundation to work on with the idea of a colorful young sidekick to Batman, inspired by the colorful Robin Hood of picturebooks), with a striking origin that gets across the point of the character so well that there have been relatively few changes to Finger's original story over the years.
Probably the biggest challenger to #38 would be Steve Ditko's last issue of Amazing Spider-Man, #38, but it is not much of a contest, really.