The purpose of this study is to develop a green and safe chitosanbased preservative which can be applied in strawberry preservation. Chitosan (CS) was treated by 2,2,6,6-tetramethylpiperidine oxygen radical/laccase oxidation system (TEMPO/laccase oxidation system), which was mainly used to prepare TEMPO/laccase chitosan (TLCS). Furthermore, on this basis, the structure and performance of TLCS were also studied. The results showed that compared with CS, the solubility of TLCS improved, and the kinetic viscosity reduced significantly. Next, a cinnamaldehyde-TEMPO/laccase chitosan (CIN-TLCS) antibacterial agent was prepared by covalently combining the aldehyde group in cinnamaldehyde (CIN) and the amino group in CS. It was found that CIN combined with TLCS through covalent bonds, which changed the structure and crystallinity of TLCS. In addition, the total antioxidant capacity of CIN-TLCS also improved, which was necessary for the application of CIN-TLCS in extending shelf life. Cytotoxicity experiments showed that CIN-TLCS had no cytotoxicity. Furthermore, strawberries were used to explore the actual bacteriostatic and fresh-keeping effects of CIN-TLCS. The experiment found that CIN-TLCS could maintain the freshness of strawberries at room temperature (23 ± 1 • C) for 5 days and had positive effects on strawberry color, loss-weight rate, hardness and pH. These results showed that CIN-TLCS could be used as a potential preserving agent for fruit storage. Practical Application: To obtain a green, safe and effective food preservative, chitosan (CS) was modified by a 2,2,6,6-tetramethylpiperidine oxygen radical/laccase oxidation system (TEMPO/laccase oxidation system) to get TEMPO/laccase chitosan (TLCS) and cinnamic aldehyde-TEMPO/laccase chitosan (CIN-TLCS). At the same time, the structure and antibacterial properties of TLCS and CIN-TLCS were analyzed, and their possibility as a new green and safe strawberry preservative was studied. Compared with oxazolidine, imidazole and triazole commercial drugs, CIN-TLCS has the advantages of low price, no pollution, no cytotoxicity and no drug resistance.