The preparation of chitosan (CTS)/microcrystalline cellulose (MCC) antimicrobial preservative film was optimized by Quadratic Rotation Combinative Design. The composite film structure was characterized by FT-IR, SEM and TG. Results showed that the optimum parameters of film formation were when chitosan solution concentration was 1.8g/100ml, MCC addition amount was 4.5g/100ml, and glycerin addition amount was 1.6g/100ml. Under this condition, the TS and elongation (E) at break of the composite film were 16.58 MPa and 17.3% respectively. Under room temperature, cherry tomatoes packed by chitosan/MCC antimicrobial preservative film (CTS-Fh) shrunk partially after 10 days, while placed under natural circumstances almost all cherry tomatoes shrunk after 4 days. Meanwhile, the composite film had excellent antibacterial activity, and the antibacterial rate (AR) for Escherichia coli and Staphylococcus aureus could be as high as 99%. Therefore, the composite film has a promising developmental prospect in the fields of fresh fruits and vegetables antibacterial preservation, biodegradable packaging film and so on.