To explore the improvement of biotransformation approach of Rosa roxburghii flavonoid aglycon releasing ability, β-glucosidase from different sources were used to hydrolyze quercetin-3-O-rutinoside, quercetin-3-O-rhamnoside, and quercetin-3-O-glucoside. Quercetin content and glycoside conversion rate as indicators, the conversion rate of quercetin glycosides and content of quercetin hydrolyzed by β-glucosidase from Lactobacillus acidophilus, Trichoderma, and almond were dynamically monitored by HPLC. And the optimized enzymatic hydrolysis time, enzymatic hydrolysis pH, enzymatic hydrolysis temperature, and enzyme dosage (enzyme-substrate mass ratio) were chose as single factors, and the effects of independent change of each factor on the index were examined. Then Box-Behnken method was used to study the influence of each factor and its interaction on the conversion rate, and to optimize the process conditions. Almond β-glucosidase hydrolyzed the three glycosides and converted them to the highest quercetin content. The conversion rates for different substrates were in the order of quercetin-3-O-glucoside(74.10%), quercetin-3-O-rutinoside(64.30%), and quercetin-3-O-rhamnoside (31.80%). The optimal optimized hydrolysis process conditions of almond β-glucosidase were hydrolysis time of 28.90min, hydrolysis pH value of 4.9, hydrolysis temperature of 52℃, and the enzyme dosage of 0.08%. Under these conditions, the conversion rate of quercetin-3-O-rutinoside, quercetin-3-O-rhamnoside, and quercetin-3-O-glucoside were 71.48%, 36.32%, and 77.86%, respectively.