Abstract: Objective：To investigate whether calculating effective degrees of freedom is necessary for evaluating the measurement uncertainty of LDH catalytic activity concentration. Methods：Firstly,E_n was calculated to verify the IFCC reference method by using LDH certificate reference material. Secondly, the catalytic activity concentrations of LDH in human mixed serum was repeatedly measured for 20 times with the reference method to calculate the degree of freedom v_A of type A uncertainty, type A relative uncertainty u_A(rel), type B relative combined uncertainty u_cB(rel) and effective degrees of freedom v_eff. In addition, the v_eff and coverage factor k_p (95% confidence level) under different ratios of u_A(rel) and u_cB(rel) were calculated. Results: When E_n was less than 1, the performance of the reference method should be excellent, and the values of v_A, u_A(rel), and u_cB(rel) were 19, 0.55%, 1.07% and 398, relatively, while therandom variables followed normal distribution. Because v_eff was more than 100, k_p was equal to 1.96 under 95% confidence level. When u_A(rel) was more than half of u_cB(rel), k_p gradually reduced with the increasing of v_A. k_p was equal to 1.96 if v_A was large enough (≥100), but the value of k_p was obviously larger than 1.96 and gradually enlarges with the increase of the ratio of u_A(rel) and u_cB(rel) if v_A was relative small. When u_A(rel) was less than or equal to half of u_cB(rel), k_p was also equal to 1.96. Conclusion: When v_A was equal to or large than 100 or u_A(rel) was less than or equal to half of u_cB(rel), there was no need to calculate v_eff. When u_A(rel) was large than half of u_cB(rel) and v_A was relative small, and k_p under 95% confidence level, the v_eff should be calculated before evaluating measurement uncertainty.