The invention relates to a preparation method of water-soluble fluorescent calcium fluoride nanoparticle. The preparation method comprises the following steps of dissolving soluble calcium salt into deionized water, so as to form a solution A; dissolving soluble citrate salt and soluble amino acid into the deionized water, so as to form a solution B; dissolving soluble fluoride salt into the deionized water, so as to form a solution C; gradually dripping the solution B into the solution A, and stirring, so as to form a mixing solution D; gradually dripping the solution C into the mixing solution D, and stirring, so as to form a mixing solution E; reacting the mixing solution E in a sealed hydrothermal reactor, and cooling to room temperature, so as to obtain a reaction solution; alternatively centrifuging and washing precipitate after centrifuging of the reaction solution by the deionized water and absolute ethyl alcohol, re-dispersing into the deionized water, and adjusting the pH (potential of hydrogen) value to 8.0 to 10.0, so as to obtain the water-soluble fluorescent calcium fluoride nanoparticle. The preparation method has the advantages that the raw materials are poisonlessand green, the sources are rich, the obtaining is easy, the cost is low, and the synthesis technology is simple; the product quality is stable, the technology repeatability is good, the crystallizingdegree and stability of the particles are good, and the water-soluble fluorescent calcium fluoride nanoparticle can be applied into biological materials and coating materials.