In order to know the role of phytoplankton communities in the distribution pattern of alkaline phosphatase activity (APA), monthly investigation was conducted in the Xiaojiang River, a tributary of the Three Gorges Reservoir (TGR). Different APA fractions (APA<sub>T</sub>, APA<sub><&thinsp;0.45&thinsp;μm</sub>, APA<sub>0.45&ndash;3&thinsp;μm</sub> and APA<sub>>&thinsp;3.0&thinsp;μm</sub>), environmental parameters, and phytoplankton communities were screened synchronously. Significant spatio-temporal differences of APA with the highest value in summer and the lowest in winter (<i>P</i>&thinsp;<&thinsp;0.05) were observed. The annual average APA<sub>T</sub> ranged from 7.78&ndash;14.03&thinsp;nmol&thinsp;L<sup>&minus;1</sup>&thinsp;min<sup>&minus;1</sup> with the highest in the midstream and the lowest in the estuary. The dominant phytoplankton species in summer and winter were Cyanophyta and Bacillariophyta, respectively. The mean cell density in the midstream and in the estuary were 5.2&thinsp;&times;&thinsp;10<sup>7</sup>&thinsp;cell&thinsp;L<sup>&minus;1</sup> and 1.4&thinsp;&times;&thinsp;10<sup>7</sup>&thinsp;cell&thinsp;L<sup>&minus;1</sup>, respectively. That APA<sub>>&thinsp;3.0&thinsp;μm</sub> were significantly higher than APA<sub>0.45&ndash;3&thinsp;μm</sub> indicated phytoplankton was the main contributor to alkaline phosphatase. Correlation analysis indicated the dominant species and cell density could determine the distribution pattern of APA. Turbidity (Turb), total phosphorus (TP), chemical oxygen demand (COD), water temperature (WT), pH and chlorophyll <i>a</i> (Chl <i>a</i>) were proved to be positively correlated with APA; soluble reactive phosphorus (SRP), conductivity (Cond), transparency (SD) and water level (WL) were negatively correlated with APA. It was concluded that spatio-temporal heterogeneity of APA determined by phytoplankton communities was related to water temperature and hydrodynamics.