Environmental issues have received increasing attention recently. Compared to solvent borne polyurethane (PU), waterborne PU requires relatively little solvent dosage. Therefore, waterborne PU is used to replace traditional solvent borne PU to satisfy environmental and economic requirements. Chitosan is a material that has been discussed widely in recent years because of its exceptional antimicrobial performance, biocompatibility, bioactivity, and biodegradability. This study primarily uses a prepolymer mixing process to synthesize m-TMXDI based waterborne PU with different ratios of aqueous chitosan and chain extenders (ethylenediamine; EDA). Modification reactions are conducted and waterborne PU modified by aqueous chitosan is synthesized. Through this experimental method, emulsions modified by different compositional proportions of aqueous chitosan to EDA were tested. For material determination, FTIR, NMR, and XPS were used to conduct chemical structure determination and GPC molecular weight tests. Regarding membrane properties, a contact angle meter analyzed the hydrophility of the membrane and the antimicrobial performance of the prepared waterborne PU membrane against Staphylococcus aureus and Escherichia coli. The test results showed the following characteristics: (1) The terminal NCO group of the waterborne PU (WPU) was bonded with the terminal amino group(-NH2) of chitosan, and (2) as the proportion of aqueous chitosan added increased, improvements were observed regarding molecular weight, hydrophility, and antimicrobial performance.