This paper investigated the ecological status of bacterioplankton in the Pearl River Estuary (PRE) through processing and analysis of 16S rRNA gene sequences obtained by high-throughput sequencing and detecting the most influenced environmental factor and the ecological niche formed by it. The results indicated that salinity was the main environmental parameters affecting the Alpha and Beta diversity of bacterioplankton in the PRE. The salinity was divided into three sequential groups: low, middle, high. The bacterioplankton in the low salinity samples had relatively high genetic diversity. The community composition analysis showed that the taxonomic information of the dominant and core OTUs of bacterioplankton under total samples and salinity groups in the study area varied widely. The dominant OTU in the total samples were Proteobacteria and the core OTUs were mostly from Acidobacteria. While the low salinity’s core OTU family were mainly C111, but the middle and high salinity samples’ were OCS111 from Acidobacteria. Moreover, the core OTUs in low salinity did not overlap with the middle and high salinity’s. Cross-grouping based OTU level on salinity and co-occurrence network analysis of the bacterioplankton showed that under the subdivided salinity grouping, OTU community formed closely related and respectively independent co-network modules, and the species composition within the modules was complex. The results showed that the salinity can effectively explain the formation of complex, closely related and independent modules of bacterioplankton, and ecological niches with specific functions, in the PRE.