She was baptized in Florence's Battistero di San Giovanni as Maria Luisa Annunziata Anna Giovanna Giuseppa Antonietta Filomena Apollonia Tommasa, in honor of her father's sister, Princess Maria Luisa (1798-1857), affectionately called "the little hunchback" by the people of Florence.

Maria Luisa was born in a time of peace and prosperity for the Grand Duchy of Tuscany, and she was raised in an loving family environment.

Later, Maria Luisa and her family were forced to flee Florence on 27 April 1859, with the outbreak of a revolution inspired by the war by France and Sardinia-Piedmont against Austria as part of the unification of Italy.

At the end of the war, her father Leopold II abdicated on 21 July and his son Ferdinand succeeded him as Grand Duke.

On 31 May 1865, at the age of twenty, Maria Luisa married Karl II, Prince of Isenburg-Birstein (1838–1899) in Brandeis, Bohemia.