Keratin particles with microscale are prepared by ball mill and its influences on the chitosan membrane is evaluated. Composite membranes with various content of keratin are fabricated, and their physical and chemical properties such as morphology, wettability, crystallization, thermal stability, tensile strength, and break elongation are investigated. Optical microscope and situ topographic scan mode of nano‐test system are used to examine the dispersion and aggregation of keratin on the surface of chitosan membrane. The result of contact angle (CA) and mechanical testing show that the incorporation of keratin particles decrease the CA from 98.1° to 58.2°. Tensile strength and break elongation of the composite membrane reaches a maximum of 65 ± 8 MPa and 15% when the keratin content is 6%, an increase of 80% and 88% compared with the pristine chitosan membrane. Both the increase in tensile strength and break elongation is result of the incorporation of keratin particles known for their excellent compatibility between keratin and chitosan matrix. These kind of composite material combines the antibacterial properties of chitosan with cell culture preference of keratin which may have potential biomedical application. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 44667.