Abstract: Objective：To evaluate the measurement uncertainty of enzyme items in medical reference laboratories by the function method. Methods：The evaluation process of the measurement uncertainty of enzyme items was divided into three parts: the identification of uncertainty sources, the quantification of uncertainty components and the establishment of uncertainty function formula. In this study, we described how to obtain the measurement uncertainty of the corresponding results by the function formula using α-amylase（α-AMY) as an example. Results：The function formula for the uncertainty of alanine aminotransferase（ALT), aspartate amino transferase（AST), alkaline phosphatase（ALP), α-AMY, γ-glutamyltransferase（GGT), creatine kinase（CK) and lactate dehydrogenase（LDH) in medical reference laboratories were u_rel=［0.000 006 6+u_rel²（reconstruct）+u_rel²（bb）+u_rel²（imprecision）］^1/2,u_rel=［0.000 006 6+u_rel²（reconstruct）+u_rel²（bb）+u_rel²（imprecision）］^1/2,u_rel=［0.000 010 2+u_rel²（reconstruct）+u_rel²（bb）+u_rel²（imprecision）］^1/2,u_rel=［0.000 008 2+u_rel²（reconstruct）u_rel²（bb）+u_rel²（imprecision）］^1/2,u_rel=［0.000 006 3+u_rel²（reconstruct）+u_rel²（bb）+u_rel²（imprecision）］^1/2,u_rel=［0.000 007 2+u_rel²（reconstruct）+u_rel²（bb）+u_rel²（imprecision）］^1/2 and u_rel=［0.000 007 2+u_rel²（reconstruct）+u_rel²（bb）+u_rel²（imprecision）］^1/2, respectively. When the concentration of α-AMY was 100U/L, the expanded uncertainty（U) calculated by the function formula was 2.5 U/L. When the concentration of α-AMY was 400 U/L, the expanded uncertainty（U) was 10.9 U/L. Conclusion：The measurement uncertainty of enzyme items in medical reference laboratories may be evaluated simply and effectively by the function method.