The community structures of periphytic algae, macroinvertebrates, and fish in the main streams and tributaries of the Weihe Basin were investigated based on aquatic ecological sampling and survey results from 77 sampling sites. An index of biotic integrity (IBI) was used to evaluate the biological integrity of the three organism groups. The results show that the health assessment results of the Weihe Basin differ with different organism groups, due to their particular responses to environmental disturbance. It was found that the assessment results based on macroinvertebrates were the best, followed by those based on periphytic algae, and the results based on fish were the worst. Generally, the health status of the aquatic ecosystem in the Weihe Basin was poor due to the high level of human disturbance. Throughout the basin, there was a tendency for the health status upstream to be better than that downstream. The main environmental variables that influence the integrity of fish, macroinvertebrates, and periphytic algae in the Weihe Basin were different from one another. Comprehensive investigation of community structure of different organism groups in the Weihe Basin will provide important scientific support for future aquatic ecosystem restoration.