Diffusion ordered NMR spectroscopy (DOSY) can be used to determine the apparent molecular weight of proteins (Mprotein) by measuring self-diffusion coefficient (Dt-protein). However, Dt-protein is also affected by the property of buffer solution, making the measurements complicated. In this work, 3-(trimethylsilyl) propane sodium sulfonate (DSS) was used as the internal reference molecule and the diffusion coefficient ratio between the target protein and DSS (Dr) was used to characterize the protein in solution. This method reduced the effects of buffer system on Mprotein determination, yielding more accurate Mprotein measurement. The Dr values of reference proteins with different molecular weight were determined. A correlation curve between Dr and Mprotein was established (lgMprotein=-2.6488 lgDr-0.7863, R2=0.997). Finally, Dr of the C-terminal domain of SARS-CoV main protease (Mpro-C) was determined and to yield its molecular weight. Accuracy and practicability of the fitting curve were demonstrated.