Fish sex ratio (SR) is an endpoint potentially indicating both endocrine activity and adversity, essential elements for identifying Endocrine Disrupting Chemicals (EDCs) as required by the EU regulations. Due to different protocols and methods in the literature studies, SR data vary greatly. This study analyses literature SR data and discusses important considerations for using SR data in the regulatory context for the hazard identification, classification, PBT (persistent, bioaccumulative and toxic) assessment, testing, and risk assessment. A total number of 106 studies were compiled for SR of zebrafish, medaka and fathead minnow exposed to 84 chemicals or mixtures. About 53% of literature studies determined SR by methods different from the standard histology method, leading to uncertainty of quantifying SR and differential sensitivity. SR was determined after depuration in 40 papers, which may lead to chemical-induced SR changes reversible to the control. SR was responsive to chemicals with EAS (estrogen, androgen, steoroidogenesis) activity and also to those with thyroid and progesterone activity. Besides, SR was influenced by non-chemical factors, e.g., inbreeding and temperature, leading to difficulty in data interpretation. The ECHA/EFSA/JRC Guidance suggests that SR and gonad histology data can be used for identifying EDCs. Due to reversibility, influence of confounding factors, and responsiveness to chemicals with endocrine activity other than EAS, this study suggests that SR/gonad histology should be combined with certain mode of action evidence for identifying EDCs. Important considerations for using SR data in the identification, classification, PBT assessment, testing, and risk assessment are discussed.