Water samplings were collected at 76 sites across Heihe River basin during the comprehensive investigation in the summer of 2009 and summer and fall of 2010.242 species, which belong to 8 divisions, 11 classes, 25 orders, 45 families and 94 genera, were identified from these water samplings. Bacillariophyta which makes up of 38.43% of the total number of the species is the dominant group, followed by Chlorophyta and Cyanophyta. Synedra ulna, Dactylococcopsis irregularis and S. acus are dominant species, whose ecological dominances are 0.060, 0.031 and 0.021. And frequencies of their occurrence are 43.42%, 43.42% and 46.05%, respectively. The analysis of biodiversity indicates that the complex and stability of phytoplankton community in middle and downstreams are both higher than those of upstream. The water quality of upstream is of no-pollution or slightly polluted, but slightly or moderately polluted in the middle and downstream. The correlation analysis between basin-wide total phytoplankton abundance and environment factors of water shows that the phytoplankton abundance is positively correlated with water hardness in the upstream while the midstream phytoplankton abundance is not significantly related with any factors. In the downstream, the phytoplankton abundance is negatively correlated with water hardness but positively correlated with nutrient index of the total nitrogen multiply by the total phosphorus (TN×TP). In the whole basin, phytoplankton abundance is positively correlated with total dissolved solids, pH and water hardness. The spatial distribution of phytoplankton in Heihe River basin shows obvious vertical zonality and horizontal zonality corresponding to differentiation characteristics of river hydrological zonation. These results showed the spatial correlationship between phytoplankton communities and their natural environment backgrounds. We hope this research could be favor to make policies to improve the management and sustainable utilization of the aquatic biological resources, to protect the water ecological environment in Heihe River basin and even other continental river basins.