Based on the data collected from the surveys in June and August 2013 in the Yellow River Estuary and its adjacent waters, the species composition, dominant species and species diversity of phytoplankton assemblage were studied, and the spatial structure of phytoplankton assemblage and its relationship with environmental variables were examined by canonical correlation analysis (CCA). A total of 88 phytoplankton species belonging to 4 phyla and 40 genera were collected, among which Bacillariophyta was the most abundant phylum, which included 30 genera and 66 species, accounting for 74.2% of total taxa, followed by Phrrophyta, which included 6 genera and 16 species, accounting for 18.2% of total taxa. The third phylum was Chlorophyta, which included 3 genera and 5 species, accounting for 5.7% of total taxa. And the least abundant phylum was Chrysophyta, which had only 1 species belonging to 1 genus. Chaetoceros curvisetus and Coscinodiscus subtilis were the dominant species in June, while the dominant species were Ceratium fusus and C. curvisetus in August. The Margalef species richness index(D), Shannon diversity index (H') and Pielou evenness index (J') of the phytoplankton community were higher in June than those in August. CCA suggested that the main factors affecting the phytoplankton community structure in June were nitrate, nitrite nitrogen, dissolved oxygen and transparency; the main factors influencing the phytoplankton community structure in August were dissolved oxygen, silicate, ammonium nitrogen and transparency. The changes in diluted water and abundant nutrients resulting from the sand-washing operation of Yellow River might the primary factors causing the difference in the spatial structure of the phytoplankton assemblage between June and August.