Water pollution has been a significant issue in the Huai River Basin (HRB) of China since the late 1970s. From December 2012, five experiments were carried out along the main streams of HRB. The monitoring indices contained physicochemical variables, habitat environmental indicators and community structure of phytoplankton, zooplankton and zoobenthos. The correlations between species diversity and physicochemical variables were analyzed using cluster analysis, correlation analysis method and redundancy analysis method. Results indicated that the species diversities of Shaying River's upstream and Huai River's mainstream were better than Shaying River's midstream and downstream. All the sections were divided into five clusters, and different clusters were affected by different physicochemical factors. Dissolved oxygen (DO), habitat quality index (HQI) and chemical oxygen demand (CODCr) were main factors affecting species diversity of Shaying River's upstream; total phosphorus (TP), total nitrogen (TN), ammonia nitrogen (NH4-N), CODCr and permanganate index (CODMn) had a great influence on the Shaying River's midstream and downstream; DO, water temperature (WT), HQI and CODCr were main factors affecting the Huai River's mainstream. These results provide valuable information for policy decision makers and stakeholders on water quality assessment, water ecosystem restoration, and sustainable watershed management in the HRB.