The special service was held at St Peter's Collegiate Church in the city centre to honour the former England international and Wolves vice president who died on November 12, aged 87.
Fans were able to light a candle and take a moment to reflect in the church, as well as sign a book of condolence.
Later, the book of condolence was moved to the Molineux reception and fans are invited to visit and pay their respects.
Wolves heroes Steve Bull and John Richards were also in attendance at the church, while the club's former head of media Paul Berry shared some words, alongside a reading from reverend David Wright.
Members of Ron's family and representatives from the club, including Scott Sellars, Matt Wild, Vinny Clark and Russell Jones were also in attendance.
Following the church service, attention then turned to Molineux as flowers were laid on the pitch pre-match, followed by a minute's applause. Both sets of players also wore black armbands.
Chants of 'there's only one Ron flowers' rang around Molineux ahead of kick-off.
Speaking in his pre-match programme notes, Wolves head coach Bruno Lage said: "I never had the privilege of meeting Ron, but I have been told stories about the impact he not only had on the club as a player, but I also know he was a real gentleman off the pitch and has a legacy which will live on around the city of Wolverhampton.
"He will be sadly missed by everyone associated with the club, and at today's match, we will pay tribute to Ron and the incredible life he led. We will do everything we can to give a performance in his honour to make you all proud."
Flowers was a star on the pitch and a popular figure in the city after his retirement, with his sports shop still trading on Queen Street.
The former midfielder is still the club's fifth highest appearance holder and was a mainstay in the side that created Wolves' most successful era under legendary manager Stan Cullis.
He won three First Division titles and the FA Cup during his 15 years at Molineux. Racking up 515 appearances in old gold between 1952 and 1967, he was also a member of England's 1966 World Cup winning squad.