This study aimed to investigate the effect and mechanism of immune enhancer chitosan oligosaccharides (COS) on non-specific immunity of E.sinensis. A total of 45 crabs (20±2) g with intact appendages and uniform individuals were selected for the experiments, and they were randomly divided into three groups, with fifteen crabs in each group. Each crab was injected with 0.1 mL of COS at concentrations of 0 (control group), 50 and 200 μg·mL^-1, respectively. Serum and blood lymphocytes were collected 24 hours later to analyze the changes in immune enzyme activity and immune genes, etc. The results showed that compared with the control group, COS treatment significantly increased the activities of superoxide dismutase, catalase, glutathione peroxidase and lysozyme in the serum of crab, significantly increased the content of nitric oxide, and decreased the content of malondialdehyde in serum. Quantitative real-time PCR results showed that compared with the control group, COS significantly increased the expression of EsToll1, EsToll2, EsMyD88, EsTube, EsPelle and EsDorsal in the Toll signaling pathway, and up-regulated the expression of anti-lipopolysaccharide factors EsALF and EsALF3. These results suggested that COS could improve the non-specific immunity and antioxidant capacity of E.sinensis, activate the Toll signaling pathway, and promote the expression of anti-lipopolysaccharide factors. This study provided theoretical support for the application of COS in E.sinensis.