The 5 June game was cancelled despite a vote in favour of it going ahead.
Out of nearly 12,000 fans, 52% were in favour and 48% against the idea, a fundraiser for the Ladies' team said.
The club said it was responding to supporters' comments and fan feedback on social media.
The Hammers are moving to the Olympic Stadium after 112 years at Upton Park.
The club said in a statement: "West Ham Ladies played under the lights at the Boleyn Ground back in March when they faced Tottenham Ladies and dialogue remains open between West Ham Ladies and the club with regards to the hosting of future fixtures."
The match, between West Ham Ladies and a West Ham 'All Stars' team, was the brainchild of Jack Sullivan, the son of West Ham co-chairman David.
West Ham Ladies Joint-Chairman Stephen Hunt said: "Obviously the Ladies are disappointed but myself and Jack Sullivan will get together over the summer to see what else we can do in the future."
West Ham beat Manchester United 3-2 on Tuesday, 10 May and then held a 45-minute celebration after full-time, involving fireworks and former players.
Eastenders actor Danny-Boy Hatchard, who plays Lee Carter in the BBC One soap opera, was among those backing the Ladies' game.
A West Ham Ladies' match has been called off so that the men's win over Manchester United can remain as the last public fixture played at Upton Park.