Abstract: In this study, SYBR Green Ⅰ fluorescence quantitative reverse transcription polymerase chain reaction and radioimmunoassay (RIA) methods were used to investigate the effects of exogenous estradiol-17β (E₂) on pituitary-thyroid axis of Carassius auratus. The study was carried out in 10 weeks during which E₂ was injected every two weeks for experiment groups at a dose of 0.5 mg per kilogram body weight (resolved in 57% ethanol), while the same dose of 57% ethanol was injected for control groups. The fish were not fed throughout the experimental period. The results showed that compared with control group, thyroid-stimulating hormone β subunit mRNA levels did not have significant changes after both 3 and 5 times of E₂ injection, but have an increasing trend. On the other hand, RIA showed that after 3 times of E₂ injection, thyroid hormone T₃ and T₄ concentration levels increased significantly compared with control group (P < 0.05); while after 5 times of E₂ injection, T₃ and T₄ levels showed extremely significant differences (P < 0.01). All of the above demonstrated that exogenous E₂ could influence the normal physiological functions of C. auratus.