Biomacromolecule has been widely used as biomedical material. Because different biomacromolecules possess different properties, how to exhibit the respective advantages of different components on one type of biomaterial becomes the hot spot in the field of biomaterial studying. This work reported a type of complex film that consisted of hyaluronic acid (HA), type I collagen (Col-I), and chitosan (CS) (HA–Col-I/CS, HCC). Then, a series of experiments were performed, such as inverted microscopic observation, atomic force microscopic (AFM) imaging, flow cytometry (FCM) measurement, MTT assay, and MIC assay. In the present work, we observed the growing condition of 3T3 fibroblasts on the surface of the HCC complex film, visualized the morphological changes of platelets during the coagulation process, and discovered microparticles on the platelet membrane. Moreover, we confirmed the microparticles are the platelet-derived microparticles (PMPs) using the FCM. In addition, the minimal inhibitory concentration (MIC) of HCC against Escherichia coli (E. coli) 8099 was 0.025 mg/ml, against Staphylococcus aureus (S. aureus) ATCC 6538 was 0.1 mg/ml. The results together indicated that the HCC film possessed promising coagulation property, cell compatibility and anti-bacteria property, and the potential in future clinical application such as wound healing and bandage.