The 422-year-old manuscript – written six hours before her execution – will be unveiled at the National Library of Scotland later this month.
Library bosses said the letter offered a once-in-a-lifetime opportunity to see a "fascinating" piece of history.
The priceless letter was written on 8 February, 1587, to Henri III, King of France, the brother of her late husband Francois II. It shows her belief that she died a religious martyr and not for purely political reasons, as the English government alleged.
And although just hours from death, the letter reveals that she even thinks of her "unfortunate" servants and asks Henri to ensure their wages are paid.
Martyn Wade, chief executive of the National Library of Scotland in Edinburgh, said: "The letter provides an incredible opportunity to see a fascinating piece of Scotland's history."
Since the letter was last on display library visitors have only been able to view a digitally-enhanced reproduction of the manuscript as it is stored under tightly-controlled conditions to prevent damage from heat, light and humidity.
For conservation reasons, the letter will be on display within a glass case for seven days only, beginning on Tuesday 15 September. It is not known when, or even if, the original will go on display again.
Mary Queen of Scots had fled to England after an unsuccessful attempt to regain the throne in Scotland.
She sought protection from her father's first cousin, Queen Elizabeth I, whose kingdom she hoped to inherit.
Queen Elizabeth I, however, ordered her arrest, because of the threat presented by Mary Queen of Scots, who was considered the rightful ruler of England by many English Catholics.
After a long period of custody in England, she was tried and executed for treason following her involvement in three plots to assassinate Elizabeth.
In the letter, written in French at 2am the night before she died, she described how she had suffered at the hands of "the Queen my cousin" for "almost twenty years" and had "finally been condemned to death by her and her estates".
She wrote: "Tonight, after dinner, I have been advised of my sentence: I am to be executed like a criminal at eight in the morning."
She added: "I scorn death and vow that I meet it innocent of any crime, even if I were their subject."
She bemoaned that she had not been able to see her chaplain, meaning she had not been given the Last Sacrament. She also notes she has been unable to make a will, in which she would have written that she wanted her body to be taken to France.
And she ended the letter by expressing her "warm feeling" for Henri III.
She told him she had sent him two precious stones, which were "talismans against illness".
"Accept them from your loving sister-in-law, who, as she dies, bears witness of her warm feeling for you," she wrote.
The letter was signed: "Wednesday, at two in the morning. Your most loving and most true sister."
The letter is being shown to tie in with the opening of the library's new 2.24 million visitor facilities.
The precious manuscript will join ten other exhibits, including a complete Gutenberg Bible, the manuscript autobiography of David Hume and some original artwork from Alasdair Gray's Lanark, which will remain on show until 8 November.
The National Library of Scotland revamp has included work on three exhibition areas, as well as a new shop and cafe.