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Reclaimed water plays an important role in alleviating the shortage of urban water resources; however, the trace pollutants and pathogens in reclaimed water have an effect on the plankton community in the receiving water. This study investigated the spatial variation mechanism of microbial community diversity in the Beijing-Tianjin-Hebei reach of the Nordkanal River based on the OTUs and phylum level fragment number and fragment abundance data matrix. The results showed that the physical and chemical disturbance caused by the frequent inflow of reclaimed water changed the hydrology and water quality of the water body, and the plankton community could be divided into two different groups along the geographical scale:the medium and upstream clustering (MUC) and the downstream clustering (DC). The analysis of diversity index based on the OTUs data matrix showed that the species diversity of the DC group was significantly higher than that of the MUC group, and the abundance distribution and evenness showed the opposite trend. The species richness was mainly determined by the fragment diversity of the occasional microflora; the evenness was mainly determined by the variation of the abundance of the dominant microflora; the sensitivity of the subcommunity structure with different abundance levels to spatial change was in the order of non-dominant microflora > occasional microflora > dominant microflora. The diversity analysis of the data matrix based on phylum level also showed that the species diversity of the DC group was significantly higher than that of the MUC group, and the change trend of abundance was the opposite; the most sensitive microflora group was the non-dominant phyla, followed by the occasional phyla, and the dominant phyla group was the least sensitive. The data matrix based on the number of level segments of the gate was more sensitive to environmental changes than the multi-degree data matrix based on the level of the gate. The environmental factors significantly related to microbial community were turbidity; permanganate index; oxidation-reduction potential (ORP); macrolide (MLs); tetracycline antibiotic (TCs); and regional response factors of salt ions, carbon, and inorganic nitrogen. In the aspect of abundance and diversity, these phylas that the DC group was significantly more than the MUC group were more significantly negatively correlated with MLs, whereas they were positively correlated with TCs, and these phylas that the MUC group was significantly more than the DC group was more significantly positively correlated with MLs. The research results can provide a theoretical basis and technical guidance for the ecological rehabilitation of urban river courses with reclaimed water as their main water supply source.