This present study described a rapid and cost-effective method for the separation and purification of natural sulforaphene from radish seeds by SP-700 macroporous resin and preparative high-performance liquid chromatography (HPLC). Sulforaphene with high purity and recovery was obtained by preparative HPLC with a C18 column and 30% methanol in ultra-pure water as the mobile phase. 12.5kg of radish seeds, which contained 87.5g of sulforaphene, produced 117.5g of sulforaphene-rich extract of 65.8% sulforaphene after primary separation by SP-700 macroporous resin. 5.9g of 96.5% sulforaphene was obtained from 9.5g of the sulforaphene-rich extract after purification by preparative HPLC. The purified compound was assessed by analytical HPLC and characterised by ESI/MS, 1 H NMR and 13 C NMR. Standard curve was developed using the purified sulforaphene to allow quantification of sulforaphene in the extracts of radish seeds by analytical HPLC.