To explore a new method for determining quercetin content in mulberry leaf, fluorescence spectrometry was used and corresponding operational parameters were discussed. The effects of ethanol volume fraction, static time, static temperature, addition of 5% Al(NO3)3 solution and pH value on fluorescence intensity of quercetin were studied with quercetin standard as reference. On the basis of single-factor experiments, significance analysis and interaction effects of various factors were optimized by response surface methodology. The results indicated that optimal detection conditions of quercetin content in mulberry leaf were ethanol concentration 100%, static time 40 min and static temperature 13 ℃. Quercetin content in mulberry leaf was measured of (41.92±0.11) mg/g and the relative standard deviation was 0.34% under above conditions. Established linear regression equation of quercetin standard curve was y=240.14x+7.0952, and the correlation coefficient was R2=0.9950 with a linear range of 0~1.5×10-2 mg/mL. This method was suitable for detection of quercetin content in mulberry leaf.