"Click" chemistry-based surface modification strategy was developed for PDMS microchips to enhance separation performance for both amino acids and proteins. Alkyne-PEG was synthesized by a conventional procedure and then "click" grafted to azido-PDMS. FTIR absorption by attenuated total reflection and contact angle measurements proved efficient grafting of alkyne-PEG onto PDMS surface. Manifest EOF regulation and stability of PEG-functionalized PDMS microchips were illustrated via EOF measurements and protein adsorption investigations. The stability of nonspecific protein adsorption resistance property was investigated up to 30 days. Separation of fluorescence-labeled amino acids and proteins was further demonstrated with high repeatability and reproducibility. Comparison of protein separation using PDMS microchips before and after surface modification suggested greatly improved electrophoretic performance of the PEG-functionalized PDMS microchips. We expect the "click" chemistry-based surface modification method to have wide applications in microseparation of proteins with long-term surface stability.