Objective: To investigate the serum levels and clinical significances of microRNA-205 (miR-205) and microRNA-221 (miR-221) in patients with colon cancer. Methods: A total of 172 patients with colon cancer (colon cancer group), 130 patients with benign diseases of colon (benign lesion group) and 70 healthy persons (control group) admitted to Central Hospital of Western Hainan from January 2016 to December 2018 were selected. The serum levels of miR-205 and miR-221 in each group were detected, and their relationships with clinicopathological characteristics of patients with colon cancer were analyzed. The diagnostic values of the serum levels of miR-205 and miR-221 in colon cancer were analyzed by receiver operating characteristic (ROC) curve. Pearson correlation analysis was used to analyze the correlation between the serum levels of miR-205 and miR-221. Results: The serum levels of miR-205 in colon cancer group, benign lesion group and control group were 2.84±0.96, 1.16±0.27 and 1.05±0.23, with a statistically significant diffe-rence (F=10.113, P<0.001). The serum levels of miR-221 in the three groups were 1.95±0.74, 0.37±0.08 and 0.32±0.05, with a statistically significant difference (F=12.416, P<0.001). Further pairwise comparisons found that the serum levels of miR-205 and miR-221 in colon cancer group were significantly higher than those in benign lesion group and control group (all P<0.001). The serum levels of miR-205 and miR-221 in patients with colon cancer were related with TNM stage (t=5.412, P<0.001; t=6.103, P<0.001), degree of tumor differentiation (t=4.573, P=0.028; t=4.805, P=0.013) and with or without lymph node metastasis (t=5.837, P<0.001; t=7.410, P<0.001). ROC curve analysis showed that the optimal cut-off values of the serum levels of miR-205 and miR-221 for the diagnosis of colon cancer were 2.17 and 1.30, respectively. The area under the curve of the two combined diagnostic method for colon cancer was the largest (0.924, 95%CI: 0.865-0.983), with the sensitivity and specificity of 91.3% and 87.4%. The correlation analysis showed that the serum levels of miR-205 and miR-221 were positively correlated in patients with colon cancer (r=0.837, P<0.001). Conclusion The serum levels of miR-205 and miR-221 are significantly increased in patients with colon cancer, and the two combined detection has a high value for the diagnosis of colon cancer.