Herein, a hybrid system consisting of ionic liquid (IL) and nonionic surfactant has been successfully developed for dynamic modification of PDMS microchips and analyte adsorption such as fluoresent dyes and proteins has been efficiently suppressed. Mutual authentication between microchip electrophoresis and confocal laser scanning microscope was carried out to characterize the multiple novel functions of the IL-containing system and a possible mechanism was raised. Soluble IL used herein not only played the role as supporting electrolyte, but also provided increased EOF in the PDMS microchannel compared with common electrolytes such as phosphate buffer. Due to the high ionic conductivity of IL, on-column field-amplified sample stacking effect was four-fold higher than that without IL. Furthermore, an excellent synergistic effect existed between IL and nonionic surfactant, which enhanced the ability of resolving analyte adsorption to PDMS surface, and was demonstrated in the sensitive and efficient determination of rhodamine B (with detection limit of 8 nM) and a well separated mixture of proteins.