In the present study, the extraction technology and preparative separation of 1-deoxynojirimycin from mulberry leaves were systematically investigated. Four extraction parameters (ethanol concentration, extraction temperature, extraction time and ratio of solvent to sample) were explored by response surface methodology (RSM). The results indicated that the maximal yield of 1-deoxynojirimycin was achieved with an ethanol concentration of 55%, extraction temperature of 80 °C, extraction time of 1.2 h and ratio of solvent to sample of 12:1. The extraction yield under these optimum conditions was found to be 256 mg/100 g dry mulberry leaves. A column packed with a selected resin was used to perform dynamic adsorption and desorption tests to optimize the separation process. The results show that the preparative separation of 1-deoxynojirimycin from mulberry leaves can be easily and effectively done by adopting 732 resin. In conclusion, 732 resin is the most appropriate for the separation of 1-deoxynojirimycin from other components in mulberry leaves extracts, and its adsorption behavior can be described with Langmuir isotherms and a two-step adsorption kinetics model. The recovery and purity of 1-deoxynojirimycin in the final product were 90.51% and 15.3%, respectively.