A novel fluorescence-based staining method was developed for phosphoprotein analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar to the mechanism of immobilized metal ion affinity chromatography (IMAC), the method employed quercetin–aluminum (III)-appended complex as a fluoroprobe to selectively visualize phosphorylated proteins among total proteins. According to the results, as low as 16–32 ng of phosphoproteins (α-casein, β-casein and phosvitin) could be selectively detected in 90 min with a wide linear dynamic range. In addition, the specificity of this novel stain for phosphoproteins was confirmed by 1-D and 2-D SDS-PAGE, dephosphorylation, western blot and liquid chromatography–mass spectrometry analysis (LC–MS/MS), respectively.