Ryan opted to watch the match with the travelling fans at Oakwell and saw Rovers collect a hard-earned point from a goalless draw.
Reports this week had already hinted at a fall-out between Ryan and the rest of the board over the protracted takeover of the club by Sequentia Capital, who Ryan had brought to the table.
The issue appears to have come to a head following a meeting between representatives of Rovers and the Irish-led consortium, which went ahead without Ryan.
Ryan's reign as chairman has coincided with the most successful era in the club's history, including four promotions, a first major cup win in the Johnstone's Paint Trophy and a return to the second tier for the first time in half a century.
He told Sky Sports News after the match: "I resigned today after 15 hard years of toil, four promotions, a cup and one relegation, so I think my record stands with anyone in the history of Doncaster Rovers, in fact probably way above most.
"I wanted to sit with the fans because that's really all I am. I have been a fan since I was seven-years-old and I go back to being a fan.
"I really enjoyed it and the reception I received, I must say, was very moving. I've never received a reception like that, the fans were absolutely incredible. So I'm really pleased - I brought them a lot of joy and a lot of success in the last 15 years, although obviously helped by many other people.
"I've resigned because I felt the club could be going backwards if we are not careful. I think maybe my presence was not entirely helpful in the sense that I brought a deal to the table which would have meant an investment in Doncaster Rovers of tens of millions of pounds and we could have had, in my opinion, a good go at the Premier League with that sort of money.
"We all signed heads of agreement but in the end it was turned down by a couple of the other shareholders.
"I improved the deal. I thought it was a great deal and then they went behind my back and went direct to the person and I thought that was a bit naughty.
"I wish them well, no hard feelings. I think they should come out of the shadows now because I've gone and let's see what they are going to do with the football club.
"I've done my bit for 15 years, let's see what they can do now and let's hope they can bring even more success to Doncaster Rovers."
Rovers issued a statement on their website after the game confirming Ryan's departure.
It read: "Everyone at the club would like to thank John for the outstanding work he has done as chairman and for his hard work and efforts through the years.
"We hope he will remain a close friend of the club and continue to show his support for Doncaster Rovers.
"Everyone at the club continues to work hard with the same ambition and drive as usual in order to achieve our aims and objectives."