Objective To evaluate the method of measuring activity of serum alkaline phosphatase(ALP) using N-methyl-D-glucamine(MEG) buffer as phosphorylation acceptor.Methods A new method for measurement of serum ALP activity was established based on reasonable organization of reagent solutions and optimization of the reaction conditions,and it was compared with the recommended methods using other buffers.Results The intra-assay CV was 0.62%(n=20) for ALP activity of 333U/L and the inter-assay CV was 2.3%(n=20) for ALP activity of 180 U/L.The range of linearity was extended to at least 2 073 U/L.The ALP activity determined by the MEG-based assay correlated well with the results determined by AMP-based assay(Y=1.29X_1 13.25,r=0.999) or DEA-based assay(Y=0.65X_2 19.19,r=0.998).The ALP reference values from healthy adults were 44 to 133 U/L with a mean value of 80.33 U/L.Conclusion MEG may be used as the phosphorylating acceptor buffer to substitute AMP or DEA for assay of serum ALP activity.