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This study was designed to optimize ethanol extraction process of unfertilized corn silk (UCS) to maximize phytochemical contents and bioactivities. The response surface methodology (RSM) with central composite design (CCD) was employed to obtain the optimal extraction conditions. The influence of ethanol concentration, extraction temperature and extraction time on total polyphenol contents, total flavonoid contents, maysin contents, 2,2-diphenyl-1- picrylhydrazyl(DPPH) radical scavenging activities and tyrosinase inhibition were analyzed. For all dependable variables, the most significant factor was ethanol concentration followed by extraction temperature and extraction time. The following optimum conditions were determined by simultaneous optimization of several responses with the Derringer’s desirability function using the numerical optimization function of the Design-Expert program: ethanol concentration 80.45%, extraction temperature 53.49°C, and extraction time 4.95 h. Under these conditions, the predicted values of total polyphenol contents, total flavonoid contents, maysin contents, DPPH radical scavenging activity and tyrosinase inhibition were 2758.74 μg GAE/g dried sample, 1520.81 μg QUE/g dried sample, 810.26 mg/100g dried sample, 56.86% and 43.49%, respectively, and the overall desirability (D) was 0.74.