Abstract: In this study, the response of zebrafish and rare minnow to 17α-ethinylestradiol(EE2) was studied using vitellogenin (Vtg)as a biomarker. Juvenile rare minnow and zebrafish were exposed to waterborne EE2 at 5ng/L,20ng/L and 100ng/L EE2 in laboratory aquaria. After the exposure of 5, 10, 15 days, the fish were sampled to measure Vtg in whole body homogenates (WBH) .The results showed that Vtg levels in juvenile rare minnow increased significantly at 5ng/L,20ng/L and 100ng/L EE2 after 5 days exposure and reached a maximal level on the day 15th. Although Vtg concentration in juvenile zebrafish increased significantly in 100 ng/L EE2 exposure group after 5 days,a significant increase of Vtg in 5ng/L and 20ng/L treatment was just found on the day 15th and on the day 10th respectively. These indicate that rare minnow is more sensitive than zebrafish in Vtg induction to 17α-ethinylestradiol. Zebrafish Vtg was identified in this study. After being separated by SDS-PAGE, zebrafish Vtg produced two protein bands with molecular weights of 140 kDa and 170kDa. Because the development of species-specific methods for its quantification has been hampered due to problems such as the small blood volume and the lack of sufficient pure antigen and specific antibodies, a polyclonal antibody raised against carp Vtg was used as first antibody to measure Vtg in whole body homogenates of zebrafish and rare minnow in this study.The results of western blotting showed that the carp Vtg antibodies cross-reacted with zebrafish Vtg and could be used to measure zebrafish Vtg.