Objective To generate transparent zebrafish, embryos may be treated with 1-phenyl 2-thiourea (PTU) to inhibit melanogenesis during embryogenesis. When working with techniques designed to study gene expression during embryogenesis, especially in teleosts, signal detection may be aggravated by pigmentation. Here we focus on the effects of PTU on embryo, and describes an optimized protocol for generating transparent zebrafish using PTU. Methods zebrafish embryos at the stage of 23 hpf(hours post fertilization) were treated with serial concentrations of PTU. Morphological and physiological endpoints were selected and quantified for unexposed and exposed embryos. Mortality, heart rate, hatching rate and SV-BA distance were detected until 80 hpf. ResultsExposure to 0.197mmol/L and 0.296 mmol/L PTU completely inhibited pigmentation while avoiding problems with embryo mortality, teratogenesis, reduced hatching frequency, and heart rate. The highest PTU concentration resulted in reduced hatching frequency and embryo mortality. We observed pericardial and yolk sac edema. Conclusion Optimized conditions can achieve complete inhibition of pigmentation for generating transparent zebrafish while avoiding toxic effects of PTU treatment.