Marine protists are essential for globally important biological processes, for instance, the biogeochemical cycles, the remineralization of organic matter, and climate regulation. Yet, it remains largely unclear which determines the diversity and distribution of active protistan communities at a regional scale. Due to the input of freshwater in the Pearl River and the intrusion of seawater, the South China Sea has significant environmental gradient changes on the spatial scale. We sampled three layers (surface, middle and deep) at eleven sites, which were relatively equidistant, along an estuary to basin transect in the northern South China Sea. The 18S rRNA amplicon sequencing method was used to investigate the diversity and distribution of protistan communities. Overall, the relative abundances of sequences and OTUs of protistan composition was dominated by Stramenopiles, followed by Alveolata. But distinct communities occupied different areas. Our results revealed that depth and environmental variables were vital factors in controlling the distinct vertical and horizontal distribution of protists. In the surface water, both total and abundant protistan communities were extremely significant correlations with geographic distance, while rare protistan communities were extremely significant correlations with environment. The rare community was the most sensitive to the environmental factors and depth, followed by abundant community, and finally total community.