The document, which could fetch between £14,000 and £18,000, is an appeal from her to the French ambassador in England to allow the safe passage of Scottish nobleman, George Douglas, to France.
It was written in Carlisle Castle two months after her escape from Lochleven Castle in Perthshire on May 2 1568, where she had been imprisoned for nearly a year following a forced abdication in favour of her infant son, James VI.
Mary hoped that by helping to ensure a safe journey to France for George Douglas, he would intercede with the French king on her behalf to help secure her freedom.
The letter will be sold live and online by fine art auctioneers Lyon & Turnbull, which has its headquarters in Edinburgh, on Wednesday February 2.
Cathy Marsden, rare books, manuscripts and maps specialist at Lyon & Turnbull, said: “It is rare for a document with Mary’s handwriting to come up for auction. Having custody of this fragile letter is quite special.
“There are 12 lines written by a secretary, and (an) additional six lines in Mary’s own hand. The letter is signed, ‘Votre bien bonne amye, Marie’, which translates as ‘Your very good friend, Mary’.
“Given Mary’s unique place in history, we anticipate a lot of interest in this sale.”
The letter asks the French ambassador to Queen Elizabeth I to lend George Douglas, the bearer, 300 ecus (gold coins) and to negotiate with the French royal family to secure George’s trouble-free passage.
Mary’s escape from Lochleven had been helped by George Douglas and his cousin, William Douglas.
She went to England to seek refuge from her cousin, Queen Elizabeth I, but was apprehended by Richard Lowther, deputy governor of Cumberland, and escorted to Carlisle Castle.
Mary was imprisoned by Queen Elizabeth I for 19 years before she was beheaded in Fotheringhay Castle in Northamptonshire on February 8 1587 at the age of 44.
Her life, marriages, lineage, alleged involvement in plots against Elizabeth, and subsequent execution established her as a divisive and highly romanticised historical character.