Abstract A simple and rapid method for multiplex protein assay based on tunable aptamer by microchip electrophoresis has been developed. Different lengths of aptamers can modulate the electrophoretic mobility of proteins, allowing the protein molecules to be effectively separated in hydroxyethyl cellulose buffer with 1.00mM magnesium ion. A non-specific DNA was exploited as an internal standard to achieve the quantitative assay and to reduce the interference. A fluorescence dye SYBR gold was exploited to improve the sensitivity and to suppress the interference from sample matrix. Under optimum conditions, quantitative assay of PDGF-BB (R2=0.9986), VEGF165 (R2=0.9909), and thrombin (R2=0.9947) were achieved with a dynamic range in the 5.00–150.0nM and RSDs in the 5.87–16.3% range. The recoveries were varied from 83.6% to 113.1%. Finally, the proposed method was successfully applied to analyze cell secretions, and then the concentration of PDGF-BB and VEGF165 were detected from 5.15nM to 2.03nM, and 3.14 to 2.53nM, respectively, indicating the established method can be used to analyze cell secretions.