Novel side chain dendronized polyurethane was synthesized by the sequential conjugation of amine-terminated fourth generation polyester dendrons with carboxyl groups at the side chain of polyurethane and deprotection of acetonide groups at the surface of dendrons. The effect of each step on the properties of the polymers was measured by GPC, DSC, SEM, AFM, and water contact angle measurement. The results showed that immobilization of polyester dendrons improve the compatibility of hard and soft segments in polyurethane and decrease the tendency of crystallinity due to the bulkiness of the substituents. After deprotection of the acetonide groups, both bulk and surface hydrophilicity of the polymer were evidently enhanced. Cell toxicity analysis showed that the side chain dendronized polyurethane synthesized in this study has exhibited excellent biocompatibility to human embryonic kidney 293T cells. The cells can effectively adhere to and proliferate on the surface of the polymer film. Overall, the present study demonstrated an efficient modification method for polyurethanes to improve their cell compatibility with high potential in biomedical applications.