The 54-year-old was cycling on a tandem bike with a 49-year-old man in Main Street, Gullane, East Lothian, when they collided with a 16-year-old cyclist travelling in the same direction at around 5.40pm on Saturday.
Both riders on the tandem fell off the bike and the woman suffered injuries to her head and pelvis.
She was initially taken to Edinburgh Royal Infirmary before being transferred to the Western General Hospital, where she remains in a serious condition.
The man suffered minor injuries and was taken to hospital before later being released.
The 16-year-old who was riding the other bike was uninjured, police said.
The road was closed for three hours while officers investigated the scene and they are now appealing for any witnesses to contact them.
Sergeant Neil Inglis said: "We are still trying to establish exactly what has happened to result in this serious injury collision and would like to hear from any motorists or members of the public who were on Main Street at the time.
"In addition, anyone with any further information relevant to this investigation is also asked to contact police immediately."