The Hyoscyamus genus has always been regarded as an important medical plant and its therapeutic effects are attributed to its contained different tropan alkaloids. Utilizing genetic engineering for production of plants belonging to this genus with more valuable pharmaceutical properties without consuming much time and cost is one of the most regarded points in this issue. Somatic embryogenesis and gathering knowledge about it is a great help. For this perpose we attempted to perform successful somatic embryogenesis, and to get more knowledge about its mechanism via assessing enzymes in different stages of embryo formation. In this study, leaves were cultured on MS medium containing BAP (1.5 mgL-1) and IAA (0.2mgL-1) lead to a successful embryogenesis. The results show that highest activity of superoxide dismutase and peroxidase had the highest activity in primary embryo-holding callus, catalase and ascorbate peroxidase had the highest activity in embryogenic callus but polyphenol oxidase had the highest activity in callus. In different stages of embryo formation from leaf explants of H. niger showed that changes of activities of these enzymes in various stages of embryo formation. The electrophoresis patterns of iso-enzymes of superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, polyphenol oxidase and auxin oxidase showed the presence of 7, 6, 1, 5, 6, 7, and 2 isosymes in these enzymes respectively. The molecular mass of the isozymes were determined and up-regulation, down-regulation, and modulation of the isosymal bands were assessed. The main as well as concervative isozymes involved in embryogenesis were also identified.