A protein-resistant surface has been constructed on the poly(methyl methacrylate) (PMMA) microfluidic chips based on a one-step modification. The copolymer of butyl methacrylate (BMA) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) is synthesized to introduce a dense PEG molecular brush-like coating on the PMMA microchannel surfaces via the anchoring effect of the hydrophobic BMA units. The PEGMA segments could produce hydrophilic domains formed on the interface so as to achieve stable electroosmotic flow, and less nonspecific adsorption toward biomolecules. The modification procedure and the properties of the poly(BMA-co-PEGMA)-coated surface have been characterized by FT-IR spectroscopy, confocal fluorescence microscopy, X-ray photoelectron spectroscopy and scanning electron microscopy. The water contact angle and electroosmotic flow of PEG-modified PMMA microchip are measured to be 36° and 5.4 × 10⁻⁴ cm² V⁻¹ s⁻¹, while those of 73° and 1.9 × 10⁻⁴ cm² V⁻¹ s⁻¹ for native one, respectively. The PEG-modified microchip has been applied for the electrophoresis separation of proteins, corresponding to the theoretical efficiencies about 16 300 and 412 300 plates m⁻¹. In the interest of achieving efficient separation while minimizing biofoulings from the serum and plasma, the fabrication of PEG-coated microfluidic chips would provide a biocompatible platform for complex biological analysis.