The flavonoids in mulberry leaves have good antioxidant activities. In this study, ultrasound-assisted extraction was used to obtain the crude flavonoids, and the macroporous resin adsorption technology was further employed to purify the flavonoids. The nonpolar resin labeled as H-103 was selected from eight kinds of macroporous resins as the adsorbent. Static and dynamic adsorption–desorption were tested to optimize the operation parameters. The experimental results show that the purity of the total flavonoids can be 36.3%, with a recovery of 83.4% under the optimum conditions. The flavonoids of rutin and isoquercitrin in the extract were detected by high-performance liquid chromatography, and their recoveries were above 80%. The devised method of isolation of the flavonoids from mulberry leaves may be used to develop a new process for medicinal purpose.