The abnormality in thyroid hormone modulation in developmental fish, vulnerable to per- and polyfluorinated substances, is of particular concerns for the alternative substances. Juvenile rare minnows, were exposed to chlorinated polyfluoroalkyl ether sulfonates (Cl-PFESAs), the novel alternatives to perfluorooctane sulfonate (PFOS), for 4 weeks followed by 12 weeks of depuration. Half lives were determined to be 33 d, 29 d, and 47 d for total Cl-PFESAs, C8 Cl-PFESA and C10 Cl-PFESA, respectively. Preliminary toxicity test suggested that Cl-PFESAs are moderately toxic to Rare minnow with a LC50 of 20.8 mg/L (nominal concentration） after 96 h of exposure. In the chronic toxicity test, fishes were exposed to Cl-PFESAs at geometric mean measured concentrations of 86.5 μg/L, 162 μg/L and 329 μg/L. In juvenile fishes exposed to Cl-PFESAs for 4 weeks, gene profile sequencing analysis identified 3313 differentially expressed genes, based on which pathways regulating thyroid hormone synthesis and steroid synthesis were enriched. Both whole body total and free 3,5,3'-triiodothyronine (T3) levels were significantly increased. mRNA expression of genes regulating thyroid hormone synthesis (corticotropin-releasing hormone (CRH), thyroid-stimulating hormone (THS), sodium/iodide symporter (NIS), thyroglobulin (TG), and thyroid peroxidase (TPO), transport (transthyretin,TTR), deiodinase (Dio1, Dio2) and receptor (TRα and TRβ) were decreased. Uridinediphosphate glucoronosyl-transferases (UGT1A) gene, regulating THs metabolism, was also decreased. In adult fish, thyroid hormone and genes expression in hypothalamic-pituitary-thyroid axis remained at disturbed levels after 12 weeks of depuration without exposure. Chronic developmental exposure to Cl-PFESAs caused persistent thyroid hormone disrupting effects in fish, highlighting a necessity of comprehensive ecological risk assessment.