Afforestation affect the surface energy balance through biophysical processes, and thus leading to the changes in land surface temperature. In recent years, with the implementation of the shelterbelt program in the Yangtze River Basin, the forest cover of the Yangtze River Basin has been increasing. However, the biophysical effect of afforestation on land surface temperature is not clear. To this end, our study used multi-source remote sensing and meteorological data, cooperating with the window search strategy and the attribution method based on the energy balance equation, to investigate the area change of afforestation and its biophysical effect on land surface temperature over the Yangtze River Basin from 2003 to 2012. Results showed that 6.71% of cropland and 1.33% of grassland were converted into forests, respectively. Compared to 2003, the forest area increased by 0.48% in 2012. Annually, conversion of cropland to forests decreased the daytime, nighttime, and daily land surface temperature by (0.26±0.03)℃, (0.08±0.03)℃, and (0.17±0.02)℃, respectively. Moreover, conversion of grassland to forests caused the cooling effect during daytime (0.77±0.13)℃ and warming effect at nighttime (0.49±0.09)℃, thus leading to the daily cooling effect (0.15±0.1)℃. Conversion of cropland to forest showed a cooling effect throughout the year, with the maximal and minimal cooling effect in June and November, respectively. Transition from grassland to forest produced the cooling effect during the most months, with the maximal and minimal cooling effect in May and November, respectively. A weak warming effect happened in December. For both two afforestation strategies, the cooling effect was stronger in the growing season than in the non-growing season. The attribution analysis of biophysical effect due to afforestation indicated that albedo and sensible heat flux dominated the warming ((0.119±0.004) ℃) and cooling effect ((-0.13±0.05) ℃), respectively, when cropland was converted to forests. For the conversion of grassland to forests, incoming longwave radiation and sensible heat flux dominated the warming ((0.903±0.166) ℃) and cooling effect ((-1.703±0.193) ℃), respectively. Furthermore, incoming shortwave radiation and emissivity produced weak cooling and warming effects, respectively, in these two afforestation strategies. The study can provide a theoretical reference for assessing the impacts of afforestation on climate and is of great practical significance for sustainable forest management over the Yangtze River Basin.