Sinigrin is a major glucosinolate present in Indian mustard (Brassica juncea L.) seeds as the precursor of the anticancer compound allyl isothiocyanate. In the present study, the adsorption and desorption characteristics of six macroporous ion-exchange resins for the separation of sinigrin from crude aqueous extracts have been compared. The results indicated that D261 resin showed the best adsorption and desorption capacity to sinigrin, and its adsorption data fit best to the Freundlich isotherm. The dynamic adsorption/desorption experiments were carried out to optimize the separation process. After treatment with D261 resin in one run, the purity of sinigrin in the product was increased 15.57-fold from 3.75% to 58.37% with the recovery of 79.82%. Meanwhile, the separation effect of D261 resin was also supported by UV and IR. The separation process using macroporous ion-exchange resin in our paper provides a novel, rapid and economical method for separation of sinigrin.