Due to intensified human activities like hydropower development, biodiversity in the upper Yellow River faces severe threats. In 2019, we conducted a survey across all reservoirs in the river section from Longyangxia to Liujiaxia in the upper Yellow River, focusing on key aquatic organisms such as benthic macroinvertebrates and fish. The benthic index of biotic integrity (B-IBI) and the fish stocking index (FSI) were used to assess the river’s biotic integrity. We collected 54 macroinvertebrate species with an average biomass of 1.28g/m², predominantly featuring species like Gammarus sp. and Palaemon modestus. Additionally, 39 fish species were captured, with Gymnocypris eckloni, Pseudorasbora parva, and Triplophysa pappenheimi being the most common, with an average weight of 43.27 g per individual. The B-IBI scores ranged from 1.06 to 3.04, averaging 78; the number of native fish species varied from 3 to 15 per location, resulting in an FSI score of 69. This indicated that the biotic integrity of the Longyangxia to Liujiaxia section was common yet still significantly impacted by human activities. This study highlights the impacts of human activities on the river’s ecosystem and aims to guide ecological protection and restoration efforts in the upper Yellow River.