Protein imprinted calcium alginate/polyacrylamide hydrogel film (CA/PAM MIP) with high toughness was prepared using bovine serum albumin (BSA) as template molecule, sodium alginate and acrylamide as functional monomers, N,N'-methylenebisacrylamide (MBAA) as the covalent cross-linker and CaCl2 as the ionic cross-linker via UV radiation-reduced polymerization. Factors affecting the adsorption capacity and imprinting efficiency of the BSA-imprinted CA/PAM hydrogel films were investigated, such as ratio of polyacrylamide/sodium alginate, film thickness, MBAA concentration and CaCl2 concentration. Results showed that the CA/PAM MIP exhibited an obvious improvement in terms of adsorption capacity for BSA compared with non-imprinted polymer (NIP). The adsorption capacity of MIP for BSA reached 22.49 mg/g, which was 2.7 times higher than NIP. The regeneration property of the BSA-imprinted CA/PAM hydrogel was distinctly improved and the imprinting efficiency of CA/PAM MIP maintained 77.95% of the initial value after five repetitions. Single and binary proteins rebinding indicated that the CA/PAM MIP exhibited good recognition performance. Cell culture experiments showed CA/PAM MIP was more suitable for cell culture than CA/PAM NIP. The residual sodium dodecyl sulfate (SDS) in the elution process leaded to the death of mouse fibroblast cells (L929) after 3 days. A moderate elution solution without residue eluent should be used to prepare MIP for cell culture. (C) 2014 Elsevier B.V. All rights reserved.