A novel molecularly imprinted polymer (MIP) as an artificial receptor for vitamin D3 is prepared and used. A sol-gel process is used to prepare the hybrid MIP. For organic part (gel) of MIP, methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA) and benzoylperoxide are used as functional monomer, cross-liker and initializer, respectively. As inorganic part (sol) of the MIP, tetraethyl orthosilicat (TEOS) is used. Main parameters affecting synthesis of organicinorganic hybrid MIP are investigated and optimized. The imprinting efficiency of MIP is evaluated by comparing the adsorbed amount of vitamin D3 by MIP with that by the related non-imprinted polymer (NIP). Betamethasone, dexamethasone, 17-beta-stradiol and 25-hydroxy vitamin D3 were chosen to study the selectivity of the MIP. This study revealed that the MIP possesses a remarkable affinity for vitamin D3. The limit of detection has been gained 1 ng/ml. The linear range and relative standard deviation (RSD %) were 1-10000 ng/mL and 4 (n = 3), respectively. The hybrid MIP provides a fast, selective and convenience separation of vitamin D3 from aqueous samples.