The invention belongs to the technical field of polymer membrane materials, and discloses a hydrophilic chiral separation polysulfone membrane, and a making method and an application thereof. The method includes the following concrete steps: dissolving 1-10 parts by weight of a functional blend additive, 3-30 parts by weight of a pore forming agent and 10-100 parts by weight of polysulfone in 100-500 parts of an organic solvent to obtain a membrane casting liquid; dumping the membrane casting liquid onto a glass plate, and striking to uniformity; allowing the obtained glass plate to stand, immersing the glass plate in a solidification bath to form a membrane, putting the membrane in water for immersion, taking out, and carrying out vacuum drying to obtain a hydrophilic polysulfone membrane; immersing the hydrophilic polysulfone membrane in a phosphate buffer solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, taking out the hydrophilic polysulfone membrane, and putting the hydrophilic polysulfone membrane in a phosphate buffer solution containing aminocyclodextrin to obtain the hydrophilic chiral separation polysulfone membrane which can be used for separating racemes.