A bioactive chromone, 8-C-glucosyl-7-O-methyl-(S)-aloesol (compound 1), was prepared by the acid-catalyzed hydrolysis of isoaloeresin D in Aloe barbadensis Miller. The hydrolysis conditions were successfully optimized by response surface methodology (RSM). The results indicated that RSM in the present study was adequate and effective. Then, reversed-phase flash chromatography was utilized for the isolation. In a one-step operation within 45 min, 26 mg of compound 1 was isolated from 153 mg of hydrolysis product, with a purity of 99.3%. These achievements would afford sufficient amount of high pure compound for chromatographic purpose and pharmacological activity screening.