A simple and sensitive fluorescent staining method for the detection of proteins in SDS-PAGE, namely IB (improved 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) stain, is described. Non-covalent hydrophobic probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid was applied as a fluorescent dye, which can bind to hydrophobic sites in proteins non-specifically. As low as 1 ng of protein band can be detected briefly by 30 min washing followed by 15 min staining without the aiding of stop or destaining step. The sensitivity of the new presented protocol is similar to that of SYPRO Ruby, which has been widely accepted in proteomic research. Comparative analysis of the MS compatibility of IB stain and SYPRO Ruby stain allowed us to address that IB stain is compatible with the downstream of protein identification by PMF.