Winstone, who will play the EastEnders and Carry On actress in her 20s, said: "I... am so excited to step into her national treasure shoes!"
The biopic will also feature two child actors as the younger Dame Barbara.
After portraying Windsor in the 1998 stage play Cleo, Camping, Emmanuelle and Dick and in TV's Cor Blimey!, Spiro reprises the role of Babs in her 50s.
Spiro said: "It's absolutely thrilling to be playing Barbara again. Tony Jordan has written a beautiful celebration of her life, but it's not your usual biopic, lots of surprises. She is so adored, so I hope we get it right and that Barbara herself loves it."
Babs follows the East End star over a 50-year period from 1943 to 1993, covering her lonely childhood and World War Two evacuation, her complicated relationship with her father, her marriages and her successful career.
Dame Barbara, who will be making a cameo in the drama, added: "I am truly thrilled to know that the BBC have secured two such talented and established actresses to portray me in their new biopic of my life. Sam Spiro has already proved herself playing me on stage and TV to great acclaim, as well as, having her own incredible career.
"Sam will now be playing me at a later time in my life which will be a new challenge for her and one she will undoubtedly achieve. And then to be lucky enough to have one of today's current dynamic actresses playing the younger me fills me with such excitement, especially knowing that it is Jaime Winstone. Boy, am I a lucky lady!"
The one-off 90-minute drama will be broadcast sometime next year.

Set two years later on from This is England '86, the two-hour drama will deal with the aftermath of the events in that four-part series.
"Just when I think my love affair with This is England is over, it pulls me back in," Meadows said.
Channel 4 has also confirmed that This is England '90, set in the rave era, is currently in development.
Picking up the action at Christmas 1988, the story will focus on the relationship between the Lol and Woody characters.
Meadows told the Stage newspaper that while Channel 4 "would have happily had me go straight to 1990, I felt I needed something in-between."
Channel 4's head of drama Camilla Campbell said: "We're beyond excited that Shane, Warp Films and the cast have agreed to return with this feature length drama.
"It is set to pull together a lot of story threads from '86, leaving the way clear for a new series of This is England '90."
This is England '86, which screened on Channel 4 last September, was named best TV drama at the South Bank Awards on Tuesday.
The original film, written and directed by Meadows in 2006, told of an impressionable young boy who becomes involved with a gang of skinheads.

Andrew Salina was found seriously injured at his home in Roystonhill at about 03:30 after emergency services were alerted to a disturbance.
He was subsequently pronounced dead at the scene by paramedics.
DCI Paul Livingstone said it appeared a weapon had been used in the "violent incident" and appealed for information about three men seen in the area.
Detectives from Police Scotland's major investigation team are treating the death as a murder. Officers were conducting door-to-door inquiries, with specialist forensic teams at the scene and additional patrols in the area.
Mr Livingstone said: "I am asking for the assistance of the local community to come forward with any information you may have. It is possible you witnessed or heard a disturbance in Roystonhill at the junction with Millburn Street at around 03:30 hours.
"We also understand that three men were seen entering the area in a small light coloured car and were seen leaving the area in the same vehicle following the alleged incident.
"I want to reassure the local community that we are doing everything we can to find out what took place and determine who is responsible for the death of Andrew Salina. Additional officers are patrolling Royston and I would ask anyone with any information or concerns to speak with them today."
Jaime Winstone and Samantha Spiro are both cast as Dame Barbara Windsor in BBC One drama Babs.
[NEXT_CONCEPT]
Writer Shane Meadows is to make a one-off Christmas special to follow on from the This is England film and TV series.
[NEXT_CONCEPT]
Police have launched a murder inquiry after a 51-year-old man was killed in his home in Glasgow.