Abstract: [Objective] To develop an evaluation method of measurement uncertainty in alkaline phosphatase （ALP） via instrumental method. [Methods] According to the detection principle of ALP and related item in Evaluation and Expression of Uncertainty in Measurement （JJF 1059.1 2012）, the study analyzed the sources of the component of uncertainty and worked out the combining uncertainty and expand uncertainty. [ Results] The measurement uncertainty of ALP was 5.89% and the expand uncertainty was 11.78% （k=2） when the confidence level was set at 95%.[Conclusion] The measurement of uncertainty can systemically and comprehensively reflect the detection result via ascertaining the involved links and determining the component of uncertainty. Based on the obtained measurement uncertainty, detection quality could be effectively improved.