Objective：To optimize urinary protein preparation method，to obtain a two-dimensional gel electrophoresis（2-DE） map with more urinary protein spots and higher resolution，and to provide technical supports for urinary proteomic researches. Methods：Firstly，urinary protein was extracted by 6 different preparation methods and then the total protein content between them were compared；secondly，protein spots as well as three-time repeated matching rate of 2-DE maps of these six methods were ana－lyzed，and P value for statistical analysis was set；finally，for the best method，2 different polyacrylamide gel electrophoresis（PAGE） concentrations and 2 different immobilized pH gradient（IPG） strips were utilized to compare protein spots and resolution of 2-DE maps. Results：Method named ultrafiltration got the most proteins（（791±17） μg，P=0.000） as well as maximum protein spots（724±29，P=0.000）. Utilization of 12.5% PAGE gel combined with IPG pH 3-10 strip was beneficial for the whole analysis of urinary proteins，es－pecially for the acid and basic proteins，however，when combined with pH 4-7 strip，a 2-DE map of higher resolution within the iso－electric point range of 4-7（P<0.05） can be obtained. Conclusion：This research establishes a 2-DE compatible urinary protein prepa－ration method and obtains more comprehensive urinary protein spots information，which lays a solid foundation for urinary proteomics research of clinical physiology and diseases.