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The American, 32, looked set to take part after warming up on the range but he then withdrew on the first tee.
The US Open champion fell on the stairs and hurt his lower back on Wednesday.
"I'm playing the best golf of my life and to have a freak accident happen yesterday afternoon, it sucks really bad," said Johnson.
"I have been worked on all morning and obviously I can take some swings, but I can't swing full, I can't make my normal swing and I didn't think there was any chance I could compete."
The 15-time PGA Tour winner added: "I was wearing socks and slipped and went down the three stairs. The left side of my lower back took the brunt of it and my left elbow is bruised as well."
Johnson's caddie was placing the ball on his tee for him on the range, while coach Butch Harmon said pain hindered Johnson's rest overnight.
Shortly before his withdrawal, he progressed from hitting wedge shots on the range to fuller swings and his involvement looked likely as he made his way to the first tee for a scheduled 19:03 BST start alongside playing partners Bubba Watson and Jimmy Walker.
Johnson was a popular pick to win the first major of the year as a result of the fine form he has shown in 2017. He has won the past three tournaments in which he has competed - February's Genesis Open, and both the WGC Mexico Championship and WGC Dell Match Play in March.
As well as winning last year's US Open by four shots, he finished ninth at the Open Championship and tied fourth at the Masters.
BBC Radio 5 live correspondent Iain Carter
Johnson took until the very last second to make what must have been an agonising decision to pull out. He was standing on the first tee before making the toughest call of his career. It is a severe blow for the player who has dominated golf this season.
He arrived here off the back of three big victories and was a justifiable favourite. All that has been lost through his freak fall at his rental home and the damage done to his back.
World number one Dustin Johnson is out of the Masters at Augusta National after suffering a back injury in a fall at his rental home on Wednesday.