Guppy (Poecilia reticulata) is an ideal model for studying environmental estrogens, and its large caudal fin has a high capacity to regenerate. This study analyzed the feasibility of caudal fin for detecting vitellogenin (Vtg), the most commonly used biomarker of environmental estrogens. Firstly, a sandwich ELISA for guppy Vtg was developed using purified lipovitellin and its antibody and it had a working range of 7.8-1000 ng/mL and detection limit of 3.1 ng/mL. The ELISA was used to detect tissue distribution of Vtg. In male guppy exposed to 50 and 100 ng/L 17β-estradiol (E2), Vtg concentration in caudal fin was higher than that in whole fish, brain, eyes, gonad, and skin, and was close to that in the liver. Furthermore, male guppies were exposed to environmental concentrations of 17a-ethinylestradiol (EE2) and bisphenol S (BPS) to validate the utility of caudal fin Vtg for detecting estrogenic activities. The lowest observed effect concentration of EE2 and BPS were lower than 2 ng/L and 1 μg/L, which were below or equal to the values reported for other species, demonstrating that caudal fin Vtg was highly sensitive to estrogenic chemicals. Therefore, caudal fins of guppies are suggested as alternative samples for Vtg biomarker detection.