The outer coat of embryo of Stenopus hispidus may affect cryoprotectant penetration and post-thaw procedures during cryopreservation studies. The mechanical method for separating embryos of S. hispidus was exhausting and time consuming. The aim of the present study was to compare the effect of different enzymatic isolation techniques to achieve the individual separation of embryos. Embryo clusters were exposed to three enzymes (trypsin, collagenase and hyaluronidase) at different concentrations (0.2–1.6 mg mL−1) for different treatment times (5–20 min). Morphological appearance and hatching competence of the embryos separated mechanically and enzymatically were assessed. The optimum conditions for the three enzymes were 10 min 0.4 mg mL−1 trypsin, 10 min 0.8 mg mL−1 collagenase and 10 min 0.8 mg mL−1 hyalruonidase and embryo hatching percentages were 43.8 ± 4.2%, 39.9 ± 4.9% and 42.2 ± 3.5% respectively. The enzymatic isolation technique does not affect either morphological appearance or hatching competence of the separated embryos. The method developed in this study will be helpful for future cryopreservation studies on S. hispidus embryos.