The famous story of brains over brawn, written by Julia Donaldson and illustrated by Axel Scheffler, was first published 20 years ago.
It has become a firm bedtime reading favourite for youngsters, having sold 14.5 million copies worldwide.
The new coin, which has been produced by the Royal Mint, will not be entering general circulation but is available to buy from its website.
A brilliant uncirculated version can be snapped up for £10, while a silver version is available for £65 and a gold Gruffalo coin is priced at £980.
The Mint has already produced another Gruffalo 50p, which launched earlier this year and shows the Gruffalo alone.
Nicola Howell, director of consumer at the Royal Mint said: "Following the fantastic response to the Gruffalo's debut appearance on a 50p coin to celebrate the 20th anniversary of the book, we are delighted to introduce an additional coin capturing Mouse and Gruffalo's first meeting, continuing the story of one of the world's most favourite literary monsters on UK coins."
Donaldson, said: "The launch of the first Gruffalo coins in February was a great 20th birthday present for him, and the issue of a second collectable 50p coin in the same year feels like the icing on his birthday cake."
Scheffler, said: "Having never imagined that the Gruffalo would appear on a coin, it is great that we have two coins in the 20th year of the book.
"A love of the story unites children across the world and I hope that they will be able to cherish these stories for a long time."
Other children's favourites which have appeared on coins include a sell-out Beatrix Potter character collection as well as Paddington and the Snowman.