Human insulin production was severely limited by its complex downstream processing. Herein, an insulinimprinted polymer with shape-memorable imprint cavities was designed for the separation and purification of insulin from complex biofluids. N-isopropylacrylamide (NIPAM) was chosen as main monomer, and acrylamide (AAm) and (3-acrylamidopropyl)trimethylammonium chloride (AAPTAC) as functional monomers. A poly(Llysine)-based peptide crosslinker, instead of the commonly used crosslinkers, was used to introduce poly(L-lysine) segments into the polymer. Because of the reversible and precise pH-induced helix-coil transition of the poly(Llysine) segments, the imprint cavities in the polymer are shape-memorable. For this reason, the insulin template can be removed completely under mild conditions, meanwhile the imprinting efficiency is significantly improved. A high adsorption capacity (595 mg/g) and a high imprinting factor (5.83) were obtained, both of which are significantly higher than the previously developed insulin-imprinted polymers. The polymer also exhibits a high selectivity towards insulin and can be used repeatedly. Using this polymer insulin spiked in human serum was selectively captured and separated, demonstrating its ability for separation and purification of insulin from complex biofluids.