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Natural and synthetic environmental estrogens (EEs) are widespread and have received extensive attention. Our previous studies demonstrated that depletion of the cytochrome P450 17a1 gene (cyp17a1) leads to all‐testis differentiation phenotype in zebrafish and common carp. In the present study, cyp17a1‐deficient zebrafish with defective estrogen biosynthesis were used for the evaluation of EEs, as assessed by monitoring vitellogenin (vtg) expression. A rapid and sensitive assessment procedure was established with the 3‐day administration of estradiol (E2), followed by examination of the transcriptional expression of vtgs in our cyp17a1‐deficient fish. Compared with the control fish, a higher E2‐mediated vtg upregulation observed in cyp17a1‐deficient zebrafish exposed to 0.1 μg/L E2 is known to be estrogen receptor–dependent and likely due to impaired in vivo estrogen biosynthesis. The more responsive vtg expression in cyp17a1‐deficient zebrafish was observed when exposed to 200 and 2000 μg/L bisphenol A (BPA) and perfluoro‐1‐octanesulfonate (PFOS). The estrogenic potentials of E2, BPA, and PFOS were compared and assessed by the feminization effect on ovarian differentiation in cyp17a1‐deficient zebrafish from 18 to 50 days postfertilization, based on which a higher sensitivity of E2 in ovarian differentiation than BPA and PFOS was concluded. Collectively, through the higher sensitivity to EEs and the capacity to distinguish chemicals with different estrogenic potentials exhibited by the all‐male cyp17a1‐deficient zebrafish with impaired estrogen biosynthesis, we demonstrated that they can be used as an excellent in vivo model for the evaluation of EEs. Environ Toxicol Chem 2024;43:1062–1074. © 2024 SETAC [ABSTRACT FROM AUTHOR]