An herbal formula, Huang-Qi-Liu-Yi Tang, is a prescription medicine that has been commonly used for the treatment of prostatitis and condyloma acuminatum over the last thousand years. In this study, response surface methodology, with a Box-Behnken design (BBD), was used to optimize the decoction conditions of an herbal formula. Astragaloside, calycosin-7-glucoside, glycyrrhizic acid, liquiritin, polysaccharide, and extractum were used as multiple evaluation markers. Soak time, water-to-medicinal herb ratio, and extraction time were determined as the three main variables by single-factor experiments and further optimized to obtain the maximum yields for the six marker compounds. Data from well-designed experiments were fitted to obtain second-order polynomial equations using multiple regression analysis, and the accuracies of these equations were evaluated and verified using statistical methods. By solving the regression equations and analyzing the three-dimensional response surfaces, optimum conditions were obtained and are summarized as follows: soak time of 57 min, water-to-medicinal herb ratio of 9:1 (mL/g), and extraction time of 48 min. Under optimized conditions, the experimental yields of astragaloside, calycosin-7-glucoside, glycyrrhizic acid, liquiritin, polysaccharide, and extractum were 0.13, 0.085, 1.64, 1.56, 72.19, and 25.64%, respectively, which was in good agreement with the values predicted by the BBD.