Stable isotope analysis is commonly used to study food web structures and material ?ows in a wide range of aquatic ecosystems. The trophic level of 44 fish and three aquatic invertebrate species was analyzed by using δ13C and δ15N analysis (Table 1). Sample collection was executed from Yichuang, Jingzhou, Shishou, Jianli, Chenglingji, Ezhou, Jiujiang and Hukou sections in the middle reaches of Yangtze River from March 2011 to December 2013. In this study, there was significant difference in the δ13C and δ15N values of fishes. The δ13C values ranged from ﹣33.83‰ （Aristichthys nobilis）﹣﹣17.36‰ （Silurus meridionalis） and the δ15N values ranged from 4.83‰ （Misgurnus anguillicaudatus）﹣15.13‰ （Culter alburnus）. The δ13C average values ranged from﹣30.06‰ （Micropercops swinhonis）﹣﹣21.24‰ （Paramisgurnus dabryanus） and the δ15N average values ranged from 6.91‰ （Mastacembelus armatus）﹣14.99‰ （Neosalanx taihuensis）(Figure 1). The trophic level of aquatic organisms in the middle reaches of Yangtze River was 2.42﹣4.88 based on the δ15N values of Bellamya purificata （5.48‰） as the baseline, concentrating primarily on the range of 2.83﹣3.61, and the average trophic level of fishes were 3.28. Trophic level more than 2.83 accounted for 80.85% of the total organisms. The least trophic levels were Mastacembelus armatus （2.42 ± 0.49） and Parabramis pekinensis （2.56 ± 0.52）. The trophic levels more than 4.0 were Neosalanx taihuensis, Coilia brachygnathus, Aristichthys nobilis and Saurogobio dumerili, the values were 4.88 ± 0.01、4.37 ± 0.27、4.32 ± 0.35 and 4.09 ± 0.78, respectively. The trophic levels values of Micropercops swinhonis, Elopichthys bambusa, Siniperca chuatsi, Culter alburnus, Mylopharyngodon piceus and Silurus meridionalis were 3.99、3.92 ± 0.16、3.89 ± 0.27、3.87 ± 0.62、3.59 ± 0.69 and 3.59 ± 0.57, respectively (Figure 2). The objectives of this study were to provide basic scientific data for the assessment and utilization of fishery resources, to provide the important reference value for further researches on the dynamic change of fishery resources’ structure and effect of human activity disturbance in the middle reaches of Yangtze River.