Antifouling modification technology is developed for many biomedical applications such as blood-contact devices and biosensors. In this work, a photo-reactive polymer containing zwitterionic carboxybetaine groups was prepared by copolymerization of two kinds of methacrylic acids with carboxybetaine and azidoaniline. The carboxybetaine moiety is for low fouling and the azidophenyl moiety is for photo-crosslinking. The synthesized copolymers were coated onto polymeric substrates, and then covalently immobilized on the substrates by exposure to UV radiation. The poly(CBMA-co-AzMA) coating revealed that cell and platelet adhesion and protein adsorption to the substrates were reduced significantly compared to the untreated substrate. Furthermore, the direct immobilization of galactosamine was carried out on the polymer coating by EDC/NHS chemistry. The galactose-immobilized surface had the potential for selecting hepatocyte adhesion from the co-population of different cell types. In addition, the incorporation of photolithographic technology could make micropatterns of poly(CBMA-co-AzMA) coating for the cell co-culture of hepatocytes and fibroblasts. This work demonstrates that the reported technique is an economic and facile tool for layers of reduced adsorption of protein modification with functional groups in one step.