Originating from the Shennongjia Forest District, Gufu River is a tributary of Xiangxi River which is the largest tributary of Three Gorges Reservoir Area in Hubei Province, central China. With the development of mining, urbanization and intensification of water source exploitation, the impacts of anthropogenic activities have gradually increased in the Gufu River Basin. Gufu River is diverse with regard to the hydrological and biological characteristics which influence the biomass of epilithic algae. However, there are no previous studies investigating the spatio-temporal variation of epilithic algal biomass and the interactions between epilithic algae and the environmental factors from the headstream of the Gufu River Basin to the Xiangxi Bay. To obtain basic biotic data of the biomass of epilithic aglae and explore the relationship between algal biomass and the environmental conditions in the stream ecosystems with substantially different human impacts, we measured the chlorophyll a of epilithic algae and the main environmental factors at 21 sites in the Gufu River and its major tributaries (Zhuyuan River) once a month from September 2010 to August 2011. From the monitored data, we analyzed the spatial and temporal distribution of chlorophyll a and the main environmental factors influencing the distribution pattern with methods of one-way ANOVA, cluster analysis, partial correlation analysis and stepwise regression analysis. The chlorophyll a concentration ranged between 0.07 mg/m² and 145.96 mg/m² with a mean of 11.63 mg/m². According to the similarity of chlorophyll a concentration in different sampling sites, all samples were divided into five groups by cluster analysis: Group 1, Group 2, Group 3, Group 4 and Group 5. The mean chlorophyll a of epilithic algae in the five groups were 5.90 mg/m², 24.51 mg/m², 23.83 mg/m², 45.59 mg/m², 50.03 mg/m² respectively. This result showed that the concentration of epilithic algal chlorophyll a had obvious spatial heterogeneity: the biomass in the upstream was lower than the downstream in Gufu River while it is contrary in Zhuyuan River tributary. This indicated that higher biomass might caused by the increased anthropogenic interference. There were significant differences in chlorophyll a concentration among various seasons: the concentration tended to be higher in winter and spring than in summer and autumn. The analysis of the relationship between the physico-chemical characteristics and the chlorophyll a concentration of the benthic algae demonstrated that chlorophyll a concentration were significantly positively correlated with total phosphorous and hardness of the water and significantly negatively correlated with water velocity. This indicated that total phosphorus in the water could be the first restriction factor of the algal growth, and water velocity has significant inhibitory effect on the algal growth. The results of the stepwise regression between chlorophyll a concentration and the environmental factors showed that water velocity, total nitrogen and dissolved oxygen were related to chlorophyll a concentration in winter and spring, while total phosphorus, water velocity, hardness, dissolved silicate and ammonia were related to chlorophyll a concentration in summer and autumn. We concluded that the spatial pattern of the algal biomass in Gufu River were determined by its habitat scale (nutrients) and watershed-scale characteristics (hardness and conductivity), while the temporal pattern of the algal biomass was mainly affected by its hydrodynamic characteristics (water velocity).