Dietary R-sulforaphane is a highly potent inducer of the Keap1/Nrf2/ARE pathway. Furthermore, sulforaphane is currently being used in clinical trials to assess its effects against different tumour processes. This study reports an efficient preparation of enantiopure R-sulforaphane based on the enzymatic hydrolysis of its natural precursor glucoraphanin. As an alternative to broccoli seeds, we have exploited Tuscan black kale seeds as a suitable source for gram-scale production of glucoraphanin. The defatted seed meal contained 5.1% (w/w) of glucoraphanin that was first isolated through an anion exchange chromatographic process, and then purified by gel filtration. The availability of glucoraphanin (purity ≈ 95%, weight basis) has allowed us to develop a novel simple hydrolytic process involving myrosinase (EC 3.2.1.147) in a biphasic system to directly produce R-sulforaphane. In a typical experiment, 1.09 g of enantiopure R-sulforaphane was obtained from 150 g of defatted Tuscan black kale seed meal.