Smart materials with both bactericidal and bacteria-resistant functions are promising for combating the infection concern of medical devices. Current work mostly utilizes hydrolysis to switch materials from antimicrobial to antifouling forms by incubating materials in aqueous solutions for hours to days. In this work, a new photoresponsive poly[2-((4,5-dimethoxy-2-nitrobenzyl)oxy)- N-(2-(methacryloyloxy)ethyl)- N, N-dimethyl-2-oxoethan-1-aminium] (polyCBNA) hydrogel was developed, incorporating the photolabile 4,5-dimethoxy-2-nitrobenzyl and cationic quaternary ammonium groups. The photolabile groups were readily cleaved from the hydrogel shortly upon UV irradiation at 365 nm (a long wavelength widely used for biomedical applications), leading to polymer surface charge switching from cationic to zwitterionic form. Protein adsorbed significantly on polyCBNA but easily desorbed from surfaces after UV irradiation. The cationic hydrogel as a precursor was shown to effectively kill the attached bacteria, and then quickly switched to zwitterionic antifouling form via photolysis, which released the attached bacteria from surfaces and prevented further bacterial attachment. Moreover, the adhered endothelial cells were easily detached from polyCBNA surfaces triggered by light, providing a facile and less destructive nonenzymatic approach to harvest cells. This smart photoresponsive polyCBNA polymer, with integrated antimicrobial and antifouling properties, holds great potential in biomedical applications such as self-sterilizing and self-cleaning coatings for implants, cell harvesting, and cell patterning.