Lactase, which can cause lactose intolerance in its deficiency, is a vital enzyme concerning digestion. To overcome lactose intolerance for patients with digestion problem depending of this kind of issue, lactose in food should be removed. In this study, lactose imprinted poly(2-hydroxyethyl methacrylate-N-methacryloyl- l -aspartic acid), poly(HEMA-MAsp), cryogels were synthesized to reduce the amount of lactose content of milk samples. Occurrence of desired bounds, structural integrity, and surface characteristics were analyzed via Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), scanning electron microscope (SEM), micro computed tomography (CT), and confocal microscope methods. Water retention characteristic were tested in solution with different electrolytic nature. Adsorption parameters were optimized in an aqueous medium. The adsorption performance of imprinted cryogels was studied in milk samples obtained from cow, sheep, goat, buffalo, and from human volunteers at different intervals after birth. Amount of lactose adsorbed in aqueous media and milk sample from humans were 322 (56.7%) and 179.5 (5.94%) mg lactose/g polymer, respectively. Selectivity studies revealed an approximately 8-fold increase in adsorption rate of molecularly imprinted cryogels as compared to that of nonimprinted cryogels. In addition, competitive adsorption was conducted using lactose-imprinted cryogels in aqueous media containing lactose, glucose, and galactose molecules resulting in adsorption rates of 220.56, 57.87, and 61.65 mg biomolecule/g polymer, respectively.