The preservation of urinary proteins on a membrane plays a vital role in biomarker research, and the efficient elution of proteins preserved on nitrocellulose membrane (NC membrane) determines the application of this method. During the heating elution procedure, we raised the temperature to reduce the intense vortexing time, and kept gentle rotating while precipitation to prevent nitrocellulose reformation. We also used SDS-PAGE and LC-MS/MS to analyze the urinary proteins prepared by heating elution procedure, intense vortexing elution procedure and acetone precipitation method. There was no degradation of proteins prepared by heating elution procedure. Compared with proteins prepared by heating elution method and acetone precipitation method, the overlapping rates of the proteins was almost the same (92.6% versus 96.8%) and the ratios of CV values (< 20%) of the proteins were both high (85.2% and 94.4%). The heating elution procedure achieved good technical reproducibility, and was much simpler and more efficient than the previous one. It can facilitate the application of the preservation of urinary proteins on membrane.