Labour's Mark Bradshaw received an email at 00:30 BST saying there was "no longer a place for you on my cabinet".
He said Mr Ferguson wanted to "get rid" of him because he is in the running to be Labour's mayoral candidate.
The mayor, who stands for re-election as an independent next year, said Mr Bradshaw was "playing party politics".
The row between the two continued on Twitter.
The Bristol Port Company bought the port from the council in 1991 and agreed a 150-year lease on the understanding it could buy the freehold at a later date.
But the council has agreed to sell it, despite opposition from councillors who had asked Mr Ferguson to reconsider his decision at a cabinet meeting on Tuesday.
The mayor told the BBC that Mr Bradshaw had "turned up at the last minute" with a prepared statement on the port sale explaining why he had "changed his mind".
He accused Mr Bradshaw of having "started to use the cabinet and his position as a political stage for his own ambition".
But Mr Bradshaw said he had been "raising questions right from the start" about the sale of the freehold.
These included where much of the Â£10m raised will be spent and "uncertainty" about whether the Henbury Loop - a rail line used for freight which campaigners hope will open for passenger use - could co-exist with an expanded port.
The Bedminster councillor said: "I think it's politically motivated. I think the mayor's been looking for a reason to get rid of me because I'm a potential challenger next year."
Mr Bradshaw's responsibilities for transport and regeneration will be taken on by fellow assistant mayor, Liberal Democrat councillor Simon Cook.
Mr Bradshaw and Marvin Rees - who lost out to Mr Ferguson in the 2012 mayoral election - are on Labour's shortlist to run for mayor next year.
Bristol mayor George Ferguson has sacked one of his cabinet councillors who opposed selling off the council's stake in the city's port.