Abstract: Bacterioplankton is an important component of aquatic ecosystems. However, their composition and function are susceptible to natural and anthropogenic stresses. To understand the effect of flood season, human life and production on the composition and function of plankton bacteria, the Qingyi River Basin in Henan Province was studied. Taking both physical geography and human factors into consideration, the river was divided into four areas: clean zone (CW), urban zone (UW), villages zone (VW), and agricultural zone (AW). By employing high-throughput sequencing technology, this study investigated the changes of bacterioplankton in the four river areas before and after flood season. The results showed that: (1) The diversity of bacterioplankton decreased during the flood season, and the diversity decreased the most in the UW area. The relative abundance of Acinetobacter and Flavobacterium decreased after the flood season. (2) There were 18 phyla and 94 genera of bacterioplanktons before and after the flood season, and there were significant differences. PCoA showed that the difference before flood season was greater than that after flood season. Further RDA revealed that total nitrogen and nitrate nitrogen were the primary environmental factors affecting the diversity of floating bacterial. (3) PICRUSt2 analysis indicated that the genes with functions of denitrification, nitrogen assimilation, and dissimilatory nitrate reduction had the highest abundance, while the abundance of the genes with nitrogen fixation and nitrification functions was low. For the genes involved in nitrogen metabolism, the abundance of 12 genes decreased during the flood season, while the abundance of other 10 genes increased. These genes were mainly involved in denitrification (nirK, norB, norC, narG, narH and nosZ), nitrogen assimilation (narB, nasA, nasB, nirB and nirA), and dissimilatory nitrate reduction (napB, napA, nrfA and nirD). The research results show that after flood season, the diversity, spatial variability and abundance of nitrogen metabolism functional genes in the bacterioplankton showed a downward trend. The most significant decreases occurred in the UW area.