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Poly(dimethylsiloxane) (PDMS)-based microfluidic systems have been gaining popularity in various applications, particularly for biological analyses because of their non-toxicity, easy fabrication, practical scalability, optical transparency, and low cost. However, because of the inherent hydrophobicity of PDMS-based material, biological samples easily and strongly interact with PDMS surfaces in biological environments, which prevents the immediate use of PDMS-based microfluidics without any surface processing. To date, various surface modification methods and different materials have been utilized to improve the repelling properties of the PDMS surface and to introduce new functional groups. Based on the recent advances in this field, we outline the main strategies utilized in PDMS surface modification in this review. We also present several applications of modified PDMS surfaces in biological analysis, such as biomolecule separation, immunoassay, cell culture, and DNA hybridization.