A chemoselective primary amine modification strategy that enables the three-component, one-pot bioconjugation is described. The specifically designed, mercaptobenzaldehyde-based bifunctional linker achieves highly selective and robust amine labeling under biocompatible conditions. This linker demonstrates wide functional group tolerance and is simple to prepare, which allowed facile payload incorporation. Finally, our studies have shown that the introduction of linker does not impair the function of modified protein such as insulin.