The objective of this study was to establish an efficient and low-cost method for preparing glucoraphenin (GRE) from stir-fried seeds of Raphanus sativus L., and then evaluate the anti-hyperlipidemia activity of GRE. The optimal resin was selected by comparing adsorption and desorption ability of six kinds of anion exchange resins for GRE. The loading and elution conditions of the optimal resin for GRE were then optimized by dynamic adsorption and desorption experiments. The results showed that D301-Cl- resin was the optimal resin, and the optimal loading conditions were the original sample solution (pH = 6) with a concentration of 0.10 g/mL, the sample loading flow rate was 2 BV/h, and the sample volume was 7 BV. The optimum elution conditions were as follows: the elution solution was 5 g/100 mL NaCl, followed by subsequent elution with 4 BV at an elution flow rate of 2 BV/h. The collected eluent was further purified by ultrafiltration and preparative high-performance liquid chromatography (pHPLC) to obtain GRE, and the molecular weight and structure of GRE were determined by high performance liquid chromatography time-of-flight mass spectrometry and nuclear magnetic resonance, respectively. The purity of GRE was detected to be 95.1% by HPLC. Animal experiments showed that GRE had anti-hyperlipidemic effects by improving serum and liver lipid levels, alleviating liver lipid deposition, and protecting the liver. This study indicated that GRE may be a potential anti-hyperlipidemia drug candidate or dietary supplement.