The row comes after a poll suggested the No campaign's lead in the referendum campaign had been cut to six percentage points.
Better Together has defended the online broadcast, which featured an actress voicing concerns about independence.
But critics claimed it was "insulting" and portrayed Scottish women as ditherers when it came to politics. The actress who appeared in the video has since been dubbed the "patronising Better Together lady".
Senior Labour sources said there was real anger over the advert among Labour women and female supporters of Better Together, just as they were trying to fight against a Yes campaign presenting itself as the alternative for women.
They said concern was centring on Mr Alexander, Labour's General Election co-ordinator, who sources say saw the advert and a similarly contentious one a week earlier before they were broadcast.
A senior party source said: "I have been inundated with angry phone calls from friends and colleagues over this week's broadcast.
"On top of the disastrous decision to include an Orange Order fanatic in the first one last week, the portrayal of Better Together as a 1950s-style Conservative and Unionist party this week was a step too far.
"Many colleagues are very angry that we have apparently gone from New Labour to old Tory and Douglas Alexander's hands are all over this.
"He has not even had the guts to come out and defend it, using females MPs and MSPs to justify this colossal error.
"Those in charge of the campaign have to rein him in and move the campaign back into a Better Together, best of both worlds, positive message portraying a modern Scotland within the UK, before it is too late".
He added: "The polls are closing, the votes are being cast and we need to convince people that we have a message about the future and not the past."
Better Together insists all the words spoken in the YouTube video were real concerns about independence which had been voiced by real women in focus groups and on doorsteps.
Hitting back at the claim it was patronising, one member of the organisation said: "What I find patronising is the idea that these concerns aren't worthy of discussion or broadcast."
Mr Alexander won praise for pulling off a coup by attracting former adviser to Barack Obama David Axelrod to work on Labour's General Election campaign.
He has also encouraged the party to do more on social media in the run-up to next year's vote.
He was also election co-ordinator for Labour's failed bid to remain in power in 2010. Later he helped to organise David Miliband's attempt to win the resulting Labour leadership contest.
Mr Alexander's office last night declined to comment.
l STV has announced the line-up for a live referendum debate on Tuesday night.
Deputy First Minister Nicola Sturgeon, Patrick Harvie MSP, co-convenor of the Scottish Greens, and actress Elaine C Smith will speak in favour of independence. Mr Alexander, Scottish Conservative leader Ruth Davidson and Labour's Scottish education spokeswoman Kezia Dugdale will represent the No camp.
The two-hour broadcast will be broadcast live with a 350-strong audience at The Assembly Rooms in Edinburgh, starting at 8pm.
Gordon Macmillan, head of news at STV, said: "This two-hour debate will give members of the public the opportunity to put their questions to key figures on both sides of the campaign. In the final days before the referendum on 18th September we are sure there will be burning questions on all the big issues."