The invention relates to a method for in-situ preparation of a modified hydroxyapatite/polyvinyl alcohol nano-composite film. The method comprises the following steps: dropping a doped calcium nitrate solution to a polyvinyl alcohol water solution under a stirring condition, and then dropping a diammonium hydrogen phosphate solution and stirring at 40-70 DEG C for 12-24h, so that the in-situ generation of a doped modified hydroxyapatite/polyvinyl alcohol solution is achieved, wherein a calcium-phosphorus ratio is at 1.67; and then adding maleic anhydride and a catalyst, conducting stirring for 4-6h, so that a mixed solution is obtained, conducting electrostatic spinning, and drying an obtained film, so that the in-situ preparation of the modified hydroxyapatite/polyvinyl alcohol nano-composite film is achieved. The method disclosed by the invention is simple to operate; the interior of the film, which is prepared through in-situ cross-linking of the doped modified hydroxyapatite which is obtained from in-situ generation, has good dispersibility and stability, and meanwhile, the film has good biocompatibility; and the film, which can be used for separating and adsorbing a plurality of proteins, bio-enzymes as well as other bio-products, has a good adsorptive selectivity on the separation of the various proteins.