A zwitterionic phosphoryldiserine (PDS)- chitosan conjugate was synthesized via Atherton-Todd reaction, and its degree of substitution of PDS and structure were characterized by 1H and 31P NMR spectra, ICP, FTIR and XRD. Thermal analysis confirmed that there existed the freezing bound water surrounding PDS-chitosan due to the introduction of zwitterionic PDS groups onto chitosan backbone. In vitro cytotoxicity and hemolysis assay demonstrated that PDS-chitosan had excellent cell compatibility. UV adsorption and fluorescence spectra revealed that the model protein, bovine serum albumin (BSA) tended to keep its native conformation and showed better thermal stability in aqueous media in the presence of PDS-chitosan. We also found that the esterase-like activity of BSA could be enhanced by low concentration of PDS-chitosan (CBSA = 0.33 mg/mL, CPDS-chitosan = 0.0625 and 0.125 mg/mL, pH = 7.4, T = 25 °C). The results indicated that zwitterionic PDS-chitosan showed great potential for maintaining protein function in biomedical applications.