Understanding the controls on seasonal variation of energy partitioning and separation between canopy and soil surface are important for qualifying the vegetation feedback to climate system. Using observed day-to-day variations of energy balance components including net radiation, sensible heat flux, latent heat flux ground heat flux, and meteorological variables combined with an energy-balanced two-source model, energy partitioning were investigated at six sites in Heihe River Basin from 2014 to 2016. Bowen ratio(β) among the six sites exhibited significant seasonal variations while showed smaller inter-annual fluctuations. All ecosystems exhibit a "U-shaped" pattern, characterized by smaller value of β in growing season, with a minimum value in July, and fluctuating day to day. During the growing season, average Bowen ratio was the highest for the alpine swamp meadow(0.60 ± 0.30), followed by the desert riparian forest Populus euphratica(0.47 ± 0.72), the alpine desert(0.46 ± 0.10), the Tamarix ramosissima desert riparian shrub ecosystem(0.33 ± 0.57), alpine meadow ecosystem(0.32 ± 0.17), and cropland ecosystem(0.27 ± 0.46). The agreement of Bowen ratio between simulated and observed values demonstrated that the two-source model is a promising tool for energy partitioning and separation between canopy and soil surface. The importance of biophysical control explains the convergence of seasonal and annual patterns of Bowen ratio for all ecosystems, and the changes in Bowen ratio showed divergence among varied ecosystems because of different physiological responses to energy flow pathways between canopy and soil surface.