Abstract: Objective：To evaluate the bias of creatinine testing by external quality assessment (EQA)and analyze the effects of calibration on the bias of tested values using Jaffe assay. Methods：The EQA data from The Clinical Laboratory Centre of Chongqing City were utilized to investigate the frozen pooledhuman serum in which the value of creatinine was determined by reference method. The differences between Jaffe assay and creatine oxidase method were analyzed and the bias was evaluated. After the routine measurement systems were calibrated using the frozen pooledhuman sera with double levels, the EQA samples were tested. Results：The number of the laboratories using Jaffe method for creatinine measurement reduced significantly in Chongqing from 2012 to 2016. The robust average values of Jaffe assay were different from enzymatic assay significantly and the difference of the values were correlated with the concentration of creatinine (r=0.774 6). The results of the investigation using frozen pooled human serum and the trueness verification program of National Center for Clinical Laboratories indicated the positive bias of Jaffe assay was observed in low levels of creatinine with maximum of 10.39% and 13.31% and the bias correlated with the concentration of creatinine (r=0.988 9), by contrast the bias was low in enzymatic assay with maximum of 1.04% and 1.49% only. After the measurement systems were calibrated by frozen pooled human sera with doublelevels, the positive bias of Jaffe assay in the low concentration of creatinine could be corrected. Conclusion：There may be a positive bias of Jaffe assay for the samples with low concentration of creatinine, which could be corrected by using the calibrators with doublelevels.