The aim of this study was to apply biochemical parameters to Onychostoma barbatulum to understand whether Wuling Farm activities have caused organic pollution from xenoestrogens to flow into surrounding streams and thereby affected area aquatic ecosystems. Individuals of the Taiwan shovel-jaw carp (O. barbatulum) were collected by fyke netting in the Cijiawan, Kaoshan, and Yusheng Rivers in the protected area of Shei-Pa National Park and in the Ikawan River outside the protected areas from 4–5 June 2015 to 20–21 September 2015. The collected male individuals of O. barbatulum were divided into two groups. In the first group monooxygenase (Mon) activity, glutathione S-transferase (GST) activity, and vitellogenin (VTG) concentration in the liver were measured right after they were captured. Those in the second group were acclimated for 14 days and then exposed to 0 (control) or 10 ng/L 17β-estradiol for 10 days and then measured for changes in Mon, GST, and VTG in their livers. The value of VTG concentrations in O. barbatulum individuals collected from the Ikawan River was 2.12 ± 1.62 μg PO42+/g protein, which was the highest among all samples, followed by those collected from the Yusheng River (0.78 ± 1.00 μg PO42+/g protein). Individuals collected from the Yusheng River had the highest liver Mon activity (4.16 ± 1.08△A650/30 min/g protein) and the highest GST activity (1.58 ± 1.13 △A340/20 min/g protein), followed by those from the Ikawan River (Mon: 3.63 ± 1.13 △A650/30 min/g protein; GST: 1.24 ± 0.73 △A340/20 min/g protein). Comprehensive analyses showed that Mon and GST activities and VTG concentrations in livers measured right after being collected were lower than individuals exposed to 10 ng 17β-estradiol/L in the laboratory. Induced VTG concentrations were 9.87 ± 0.89 μg PO42+/g protein, Mon activity 8.02 ± 1.74 △A650/30 min/g protein, and GST activity 3.24 ± 0.62 △A340/20 min/g protein, indicating that farming activities have not significantly affected these aquatic organisms. However, pollution sources are still releasing pollutants containing xenoestrogens into the Yusheng River and Ikawan River. The impact of xenoestrogens is worthy of continuous long-term follow-up monitoring.