Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disease in women of childbearing age, with an incidence of 5–10%. This study compared the traits of zebrafish with three diagnostic criteria for human PCOS, and the diagnostic criteria for zebrafish PCOS were proposed: decreased fecundity, elevated testosterone (T) or 11-ketotestosterone (11-KT) levels and increased cortical-alveolar oocyte (CO) ratio, enhancing the zebrafish PCOS model's accuracy. According to the mammalian PCOS classification, the type of zebrafsh PCOS is divided into four phenotypes (A, B, C and D), but the four phenotypes of zebrafish PCOS are not fully covered in the existing studies (A and D). In this study, we successfully induced phenotype B zebrafish PCOS model using the aromatase inhibitor, letrozole (LET). That is, wild-type female zebrafish were exposed to 1000 μg/L LET for 30 days. Reproductive tests showed decreased fecundity in female zebrafish exposed to LET (Control: 132.63, 146.00, 173.00; LET: 29.20, 90.00, 82.71). Hormone analysis showed that female zebrafish exposed to LET had significantly lower 17β-estradiol/testosterone (E2/T) ratios, indicating elevated T levels. Meanwhile, levels of 11-KT in the ovaries exposed to LET were significantly up-regulated (Control: 0.0076 pg/μg; LET: 0.0138 pg/μg). Pathological sections of the ovary showed fewer CO in the LET-exposed group (Control: 16.27%; LET: 8.38%). In summary, the zebrafish PCOS model summarized and studied in this study provide a reliable and economical tool for the screening of therapeutic drugs, as well as for the etiology research and treatment strategies of PCOS.