To further understand the migration and transformation characteristics of phosphorus and its relationship with eutrophication in the Three Gorges Reservoir, the physical and chemical parameters, alkaline phosphatase activity of 12 typical tributaries of the reservoir were analyzed from July 2014 to July 2015. The research found significant seasonal and regional variations of alkaline phosphatase activity in the tributaries, and the heterogeneity was closely related to the degree of eutrophication. In temporal view, alkaline phosphatase activity was gradually increased from spring to summit in summer. The activity of alkaline phosphatase then dropped from autumn to bottom in winter. On the spatial side, the activity of alkaline phosphatase had high values in Xiaojiang River, Xiangxi River and Zhuxi River, which were at high level of eutrophication. The activity of alkaline phosphatase had low values in Daning River and Shennong River, which were at low level of eutrophication. The correlation analysis results showed that there was a significant positive correlation between alkaline phosphatase activity and water temperature, dissolved oxygen, conductivity, pH and turbidity; there was a significant negative correlation between alkaline phosphatase activity and transparency in each tributary. In terms of nutrient, alkaline phosphatase activity was significantly negatively correlated with soluble active phosphorus and positive correlated with chemical oxygen demand, chlorophyll-a and trophic state index. The contributions of different organisms and the influences of different environmental factors are the main causes of the spatial and temporal heterogeneity of alkaline phosphatase.