James Gorin von Grozny, from Devon, paid £150 for the work which he believes was painted by Sir Edwin Landseer in 1838.
But art experts say Landseer would have had no call to paint the sisters who were not famous at that date.
The only known portrait of the sisters was painted by their brother, Branwell.
In the painting, the figure believed to be Emily Bronte holds a pen and notebook, whilst Charlotte stands and Anne looks away to one side.
Mr Gorin von Gronzy had originally bought a different picture of three sisters from an auction house, but when he went to collect it, it had disappeared.
He said the auction house offered him a refund, or the picture he now believes is of the Bronte sisters.
Professor Francis O' Gorman, of the University of Leeds, an expert in Victorian Literature, said he was doubtful that the painting depicted the Bronte sisters.
"The Brontes were unheard of outside their family circle in 1838.
"There was nothing in the public domain which might have attracted one of the most famous painters of the early Victorian period to stop by and paint them", he said.
However, Mr Gorin von Grozny said that Landseer could have travelled through the Brontes' home town of Haworth whilst visiting his friend John Nussey at Bolton Hall in Yorkshire.
Nussey was the also brother of Charlotte Bronte's friend Ellen.
Mr Gorin von Gronzy said a small 'EL' monogram and the date 1838 visible in the crook of 'Charlotte's' arm, led to his belief that Landseer was the artist.
It is thought that the key to the painting's authenticity could lay in a sketch of a knee on the back of the portrait.
The sketch apparently shows a leg with a three inch scar just below the knee.
Mr Gorin von Grozny argued that a painting by Charlotte Bronte depicting a shepherdess, apparently with a similar scar on her leg, could have been a self-portrait.
The painting of the shepherdess by Bronte, based on Solitude at Dawn by Johann Henry Fuseli, appeared in a book called The Art of the Brontes.
And that would all be really exciting if there weren't big, huge 'but's to everything. And there's practically no need to write the arguments as a quick look at the picture clearly tells that these are not the Brontë sisters. Being three and holding a pen - when they were nearly 10 years away from becoming published authors - is not enough: the dresses, the faces, etc. seem to be all wrong, not to mention that the Edward Landseer - John Nussey - Ellen Nussey - Charlotte Brontë theory is a bit tenuous to put it mildly.