This study employed metagenomic sequencing technology and multivariate statistical methods to systematically analyze the spatial distributions and environmental driving factors of eukaryotic microbial communities, as well as their nitrogen metabolism genes at 15 sites along the Ba River. The results indicated significant differences in the α diversity of eukaryotic microbes among the upper, middle and lower reaches, with higher diversity in the lower reaches rather than that in the upper reaches. In terms of community composition, Bacillariophyta (48.3%) was the dominant phylum, and the relative abundance of Ciliophora remarkably increased in the middle and lower reaches. Nitrogen metabolism in eukaryotes was dominated by assimilation in the upper reaches, with a gradual increase in the abundance of transport-related genes in the middle reaches, and denitrification intensified in the lower reaches. Furthermore, TP, NO₃^⁻-N and NH₄^⁺-N significantly affected the eukaryotic microbial community structure, while NH₄⁺-N, NO₃^⁻-N, Chl a, COND and salinity were the key factors driving the distribution of nitrogen metabolism genes.