The invention relates to a preparation method of glucose vinyl ester/isopropyl acrylamide copolymer nanofiber membrane. The preparation method provided by the invention comprises the following steps of: dissolving adipic acid divinyl ester and glucose into anhydrous pyridine, adding alkaline protease, reacting, separating and purifying to obtain glucose ethylene ester; adding N-isopropyl acrylamide, an initiating agent and a solvent into the glucose ethylene ester, then stirring and reacting under the protection of nitrogen, and carrying out polymerization reaction to obtain glucose ethylene ester/isopropyl acrylamide copolymer; and adding the glucose ethylene ester/isopropyl acrylamide copolymer and PLCL into the solvent, stirring to obtain a spinning solution, then carrying out electrostatic spinning, and drying, so that the glucose vinyl ester/isopropyl acrylamide copolymer nanofiber membrane is obtained. The preparation method is simple in operation, products are easy to treat, and the preparation method is economic and environment-friendly; and the nanofiber membrane contains abundant reactive hydrophilic activity functional groups and has a phase transition temperature close to body temperature, so that specific adsorption on surface of material is enhanced while biocompatibility on the surface of the material is improved and multiple potential applications can be realized on the basis of phase transition properties.