Heritage Auctions will put the John Romita Sr artwork up for sale later this month, reports Auction Central News.
The cover features Spider-Man fighting Kraven the Hunter and the Vulture.
"This cover was only John Romita Sr's 11th for the title and it's simply one of his very best," said Ed Jaster, senior vice president at Heritage Auctions.
"It's not just a cool cover; it's a priceless piece of Silver Age Marvel lore and only the second Romita Sr Spider-Man cover from this era we have offered to date. We're expecting some fireworks when this comes up."
Heritage has various other comics-related items coming up for sale this month, including Victor Moscoso's original treatment for the Zap Comix #4 wraparound cover, Steve Ditko's original artwork for page 17 of The Amazing Spider-Man #12, a page of artwork from an unpublished Superman story by Joe Shuster, Paul Cassidy and Wayne Boring, and issues #28, #39, #40 and #50 of Amazing Spider-Man.
Amazing Spider-Man #49 was originally published in June 1967, and was written by Spider-Man creator Stan Lee with interior art by Romita Sr.
A copy of X-Men #1 was sold for $200,000 (£122,000) in a private sale earlier this year.