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Fluorescence polarization (FP) assays are widely used to quantify biomolecules, and their combination with microfluidic devices has the potential for application in onsite analysis. However, the hydrophobic surface of polydimethylsiloxane (PDMS)-based microfluidic devices and the amphiphilicity of the blocking agents can cause the nonspecific adsorption of biomolecules, which in turn reduces the sensitivity of the FP assay. To address this, we demonstrated an FP assay with improved sensitivity in microfluidic devices using a polyethylene glycol-based surface modification to avoid the use of blocking agents. We evaluated the effectiveness of the modification in inhibiting nonspecific protein adsorption and demonstrated the improved sensitivity of the FP immunoassay (FPIA). Our study addressed the lack of sensitivity of FP assays in microfluidic devices, particularly for the quantification of low-abundance analytes. [ABSTRACT FROM AUTHOR]