Abstract: Triplophysa tenuis plays an important role in maintaining the flow of material and energy in aquatic ecosystem. In this study, we applied fatty acid biomarker method and carbon and nitrogen stable isotope technique to study the feeding and nutritional ecological niche characteristics of T. tenuis. The results show that a total of 22 fatty acids were detected in the muscle, including eight kinds of saturated fatty acids (SFA), six kinds of monounsaturated fatty acids (MUFA) and eight kinds of polyunsaturated fatty acids (PUFA). The specific fatty acid analysis reveals that T. tenuis feeds on zooplankton, diatoms, land plants, or fish and shrimps that like to feed on diatoms. The carbon stable isotope (δ¹³C) values and nitrogen stable isotope (δ¹⁵N) values ranged from −27.09‰ to −20.98‰ and from 5.71‰ to 8.45‰, while trophic levels ranged from 2.68 to 3.48. There were no significant differences between males and females (P>0.05) in δ¹³C value, δ¹⁵N value or trophic level. Males had higher standard ellipse area (SEAc) and total area of convex hull (TA) than females. There were significant changes in δ¹³C values with increasing body length (P<0.01), and insignificant changes in δ¹⁵N values and trophic level. The SEAc areas of 70−80 mm body length group did not overlap with those of greater than 90 mm body length group, and were categorized into different groups in the cluster analysis. In conclusion, T. tenuis is omnivorous and its food composition varies with body length As a predator with high trophic level, it can extend the food length chain length and increase the complexity of food web, which is conducive to the maintenance of stability of aquatic ecosystem.