Mastering the characteristics of planktonic microbial communities under the influence of basin-scale environmental factors can provide scientific basis to develop more targeted protection and restoration measures. Here, an environmental DNA metabarcoding technology was applied to investigate the phytoplankton and microzooplankton simultaneously along the whole basin of Jialing River, one of the most important rivers in Southwest China with large scale altitude gradient and water temperature discrepancy. Along the river-way from upper to lower reach, an upward trend in richness of both phytoplankton and microzooplankton was observed with significant positive and negative correlations to water temperature and altitude (Spearman correlation, p < 0.05), respectively. Significant variations in their compositions along the river were also uncovered with Chrysophyta and Foraminifera was potential keystones recognized by the co-occurrence network analysis. Additionally, exponential distance-decay of similarities were detected for both phytoplankton and microzooplankton, which were stronger for phytoplankton. Based on the results of variation partitioning analysis and null model, variations in phytoplankton communities were more governed by environmental selection than those in microzooplankton. Furthermore, the co-occurrence network showed a declining trend in the complexity and stability from the upper to lower reaches. These findings could serve to establish a basis for the investigation of phytoplankton and microzooplankton communities in rivers with multiple artificial modifications.