Abstract: Zwitterionic polymers are those that have both cationic and anionic charged moieties on the same side chain maintaining overall charge neutrality. Due to their ultra-hydrophilicity via electrostatically induced hydration and excellent biocompatibility, zwitterionic polymers have been extensively used in biomedical research. In this review, we first give a brief introduction to the physico-chemical properties, classification and synthesis of zwitterionic polymers. Specifically, we summarize the recent advances of zwitterionic polymers in the following fields. (1) Antifouling coatings: Unlike traditional antifouling polymers, zwitterionic polymers generate a tightly bound and structured water layer around the zwitterionic head groups which offer better antifouling properties in complex environments. Moreover, the surface modification strategies are also discussed. (2) Modification of proteins: Zwitterionic polymers have been applied to the modification of various kinds of proteins to increase the solubility and stability, inhibit the aggregation, enhance pharmacokinetics, reduce immunogenicity and mitigate the bioactivity loss of proteins. (3) Drug delivery system: Due to their excellent hydrophilicity, biocompatibility and nonfouling properties, zwitterionic polymers have been used to increase the solubility and stability of drug carrier, extend the circulation time, and enhance the cellular internalization. (4) Membrane separation system: The fabrication and function of zwitterionic polymers in separation membrane are briefly discussed. Finally, we conclude with a perspective for the development of zwitterionic polymers in the future.