〔Abstract〕 Objective To analyze the interference of hemolysis on routine biochemical project tests. Methods 120 blood samples collected from the outpatient physical examination center of Chongqing Shuangqiao Economic and Technological Development Zone People's Hospital from April 2019 to April 2020 for this study. All subjects' blood samples were placed in two tube, respectively, and labeled as tube 1 and tube 2. Samples from tube 1 were subjected to manual hemolysis, and routine biochemical project tests were analyzed for blood samples from tube 1 and tube 2. Results There was no significant effect on blood glucose (GLU), triacylglycerol (TG), creatinine (Cr), and blood urea nitrogen (BUN), and the difference was not statistically significant (P > 0.05). The hemolysis affected cholesterol (CHO), albumin (ALB), acid dehydrogenase (LDH), aspartate transaminase (AST), alanine transaminase (ALT), direct bilirubin (DBIL), and total bilirubin (TBIL), with significant differences (P < 0.05). Conclusion The hemolysis of blood samples will affect the accuracy of the results of many routine biochemical test items. The staff should actively analyze the causes of the hemolysis in blood samples, and also take relevant measures to avoid this situation.