This study presents the development of a cholesterol-selective adsorbent that can be easily produced for the highly efficient removal of cholesterol from milk. A fundamental affinity separation technology which was combined with the specific recognition property of molecular imprinting with a high flow rate and the resulting cryogel was used to separate cholesterol separation from milk samples. The proposed material offers a reasonable pore size and structure, high surface area, and mechanical and chemical stability. To separation the cholesterol from milk, poly(2-hyroxyethyl methacrylate-N-methacryloyl-c-tryptophan methylester) cryogel beads were prepared using a functional monomer that allowed the formation of cholesterol-selective binding sites and enhanced the selective removal of cholesterol from milk. Characterization studies of the cholesterol-imprinted cryogel beads (CHO-MIPs) were carried out by attenuated total reflectance-Fourier transform infrared spectroscopy, scanning electron microscopy, elemental analysis, water-uptake tests and surface area measurements. The interactions between CHO-MIP and cholesterol were investigated and the factors affecting the adsorption of cholesterol were determined to find optimum conditions. Reusability as a measure of the continuity of the prepared CHO-MIPs was also investigated. The selectivity of the CHO-MIP beads was determined by using competing molecules (estradiol and progesterone), which are cholesterol analogues. The experimental data showed that the specific areas of the CHO-MIP and non-imprinted (NIP) cryogel beads were 17.6 and 14.7 m(2)/g, respectively. The CHO-MIP cryogel beads were 4.77 and 2.76 fold more selective for cholesterol compared to estradiol and progesterone respectively. The cholesterol adsorption capacity of the CHO-MIP beads was 288.72 mg/g when the cholesterol concentration in solution was 3.0 mg/mL. After eight adsorption-desorption cycles, the adsorption capacity of the CHO-MIP beads decreased by 9.21 %. The Langmuir-Freundlich isotherm model was well fitted as compare to Langmuir and Freundlich isotherms. The obtained kinetics data showed that a pseudo-second order mechanism was predominant for the CHO-MIP cryogel bead adsorption.