The moving note urges the world to remember victims of landline disasters and was written just two weeks before she died in a car crash on August 31 1997.
It was penned after a trip to Bosnia where she met survivors of landmine injuries as part of her charity work which was of vital importance to her.
It was written hours before Diana began a summer break which saw her and boyfriend Dodi Fayed die in the French capital while being pursued by paparazzi.
In her letter to fellow campaigner Dilys Cheetham, Diana revealed her trip to Bosnia had "strengthened her resolve" to spread the message about landmine victims.
I could not help but be deeply moved by the experience
She wrote: "I could not help but be deeply moved by the experience which hardened my resolve to ensure that the world does not forget that those who have been so needlessly maimed by these terrible weapons will need care and support for many years to come."
The letter is dated August 11 1997 and is written on Kensington Palace headed paper and is signed off "With love from Diana".
It was put up for sale by landline campaigner Mrs Cheetham in a charity auction in 1999 and bought by celebrity photographer Jason Fraser.
Fraser, who knew Diana personally, sold the letter in 2007 during the 10th anniversary of her death and it was bought by a private British collector.
It is expected to fetch £3,000 when it is sold at auction later this month.
Mark Huddleston, from Birmingham auction house Fellows, said: "The lady to whom the letter is written to, Dilys Cheetham, was a landmine campaigner who had told Diana of her own work in Bosnia.
"Following a trip to Bosnia in August 1997, Diana wrote to Dilys to thank her for her work helping refugees in the Mostar region of the country.
"She said the trip had strengthened her resolve to make sure the world did not forget those injured by mines.
"Just two weeks after she sent the letter she was dead.
"The letter is moving not only because of its heartfelt message but also because it would have been the last ever written by Diana.
"It is an incredibly important historical document.
"Princess Di was very much loved not just in Britain but around the world, and even 17 years after her death she is still very much in people's consciences."
The auction will take place on April 28.