Defatted seed meals, the byproducts of Indian mustard seed oil industry, which are just used as animal feedstock and nitrogen fertilizers, possess various nutrients and phytochemical compounds, such as sinigrin and gluconapin. The present study demonstrated a novel, low-cost, and efficient method for the co-production of sinigrin and gluconapin from defatted Indian mustard seed meals by D261 strong basic anion-exchange macroporous resin and medium pressure liquid chromatography (MPLC). The adsorption/desorption capacities of eight adsorbents characterized by BET, IR, and EDS were screened. 26.4 g defatted Indian mustard seed meals, which contained about 1210.3 mg sinigrin and 696.6 mg gluconapin, could produce 2059.6 mg of glucosinolate-rich extract of 53.67% sinigrin and 31.30% gluconapin after separation by D261 resin. Then, 949.40 mg of 97% sinigrin and 568.40 mg of 95% gluconapin could be obtained after the extracts were purified by MPLC, with the recovery of 76.03% of sinigrin and 77.38% of gluconapin. The products were assessed by analytical HPLC and characterized by UV, MS, H-1 NMR, and C-13 NMR. In conclusion, this method is practical and environmentally friendly, and it is a low-cost process to make full use of Indian mustard seeds.