Objective To investigate and analyze the clinical application value of biochemical tests in the diagnosis of diabetes mellitus. Methods A total of 4616 subjects from January 2016- September 2016 in our hospital for biochemical test were selected and randomly divided into group A and group B, 2308 subjects in each group. The fasting blood glucose test was performed in group A, while the glycosylated hemoglobin test was performed in group B, and the results were analyzed and compared. Results There was no significant difference between the two groups in the correct diagnosis rate of diabetes mellitus (P>0.05). Glycosylated hemoglobin and fasting blood glucose levels in diabetic patients were higher than those of normal subjects, and the difference was statistically significant (P<0.05). Conclusion In the diagnosis of diabetes, glycosylated hemoglobin has a higher application value than fasting glucose test, with simple operation, fast of test results and high coincidence rate, which can provide the basis for the diagnosis and treatment of diabetes, it is worthy of clinical application.