Objective To analyze the expression and clinical significance of serum exosome miR-320 in patients with colon cancer.Methods A total of 50 patients with colon cancer admitted in our hospital between April 2018 and April 2020 were included in the study, and 50 healthy patients were included in the control group. The serum samples were collected from the two groups, and the expression of miR-320 in serum exosomes was detected by RT-qPCR method, and the diagnostic value of colon cancer was analyzed.Results There were a large number of exosome particles in the purified serum samples, and its characteristics were consistent with those reported in other relevant research. The relative expression of miR-320 in serum exosomes of patients with colon cancer was significantly lower than that of healthy controls [(0.69±0.23) vs. (1.53±0.48), t=11.16, P<0.001]. The area under the curve for serum exosome miR-320 in the diagnosis of colon cancer was 0.871 (95% CI: 0.596~0.925), with the diagnostic sensitivity of 90.73%, and the specificity of 82.43%.Conclusion The expression of miR-320 was down-regulated in the serum exosomes of patients with colon cancer. The miR-320 in serum exosome may become an evaluation indicator for early diagnosis of colon cancer.