In this paper, titanium dioxide nanoparticles (TiO2 NPs) were employed to construct a functional film on PDMS microfluidic channel surface, which was formed by sequentially immobilizing poly(diallyldimethylammonium chloride) and TiO2 NPs on PDMS surface by layer-by-layer assembly technique. The modified PDMS microchip exhibited a decreased and stable EOF, which was favorable for the separation of biomolecules with similar migration times. Arginine, phenylalanine, serine and threonine were used as model analytes to evaluate the performance of the modified microchip. The four amino acids were efficiently separated within 100 s in a 3.7 cm. long separation channel and successfully detected on the carbon fiber electrode in conjunction with in-channel indirect amperometry. Resolutions and theoretical plate numbers of the analytes were considerably enhanced in the presence of TiO2 NPs. The modified microchip demonstrated excellent stability and reproducibility with improved RS Ds of migration times and peak currents for run-to-rum, day-to-day and chip-to-chip analyses, respectively. Variables influencing the separation efficiency and amperometric response, including injection and separation voltage, the working electrode position and buffer concentration, were optimized in detail.