Abstract: Objective：To establish a reference method of isotope dilution gas chromatography tandem mass spectrometry（ID-GC/MS) for the determination of human serum urea（Urea), and apply it in the truenessbased proficiency testing for assigning target values of fresh frozen serum samples. Methods ：The Urea reference method was established based on the recommended method by the Joint Committee for Traceability in Laboratory Medicine. The precision and accuracy of the established reference method were evaluated by the standard reference material（SRM) 909c from the National Institute of Standards and Technology and 2014 RELA-A and 2014 RELA-B samples provided by the International Federation of Clinical Chemistry, respectively. Then, the established reference method was used to assign the target values of fresh frozen serum Urea in the small molecule truenessbased proficiency testing of Shanghai region. Results：The relative bias of SRM 909c determined by the established reference method was -0.25％, and those of 2014 RELAA and 2014 RELAB were 0.00% and -0.10%, respectively. The imprecisions of the established reference method were less than 1.5%. When the average from all laboratories was set as the target value and the allowable total error of 8% was as the standard, the unqualification rates of samples for 201411, 201412, 201511 and 201512 were 4.17%, 1.39%, 0.00% and 1.59%, respectively. If the value assigned by the reference method was set as the target value, the unqualification rates of samples for 201411, 201412, 201511 and 201512 were 11.11%, 1.39%, 1.59% and 3.17%, respectively. Conclusion：The established reference method of ID-GC/MS for the determination of serum Urea has good precision and accuracy, which may be used in the truenessbased proficiency testing of clinical laboratories in Shanghai region.