The numerous lakes on the Tibetan Plateau play an important role in the regional hydrological cycle and water resources, but systematic observations of the lake water balance are scarce on the Tibetan Plateau. Here, we present a detailed study on the water cycle of Cona Lake, at the headwaters of the Nujiang-Salween River, based on 3 years (2011–2013) of observations of δ18O and δ2H, including samples from precipitation, lake water, and outlet surface water. Short-term atmospheric water vapor was also sampled for isotope analyses. The δ2H-δ18O relationship in lake water (δ2H = 6.67δ18O − 20.37) differed from that of local precipitation (δ2H = 8.29δ18O + 12.50), and the deuterium excess (d-excess) in the lake water (−7.5‰) was significantly lower than in local precipitation (10.7‰), indicating an evaporative isotope enrichment in lake water. The ratio of evaporation to inflow (E/I) of the lake water was calculated using both d-excess and δ18O. The E/I ratios of Cona lake ranged from 0.24 to 0.27 during the 3 years. Observations of atmospheric water vapor isotopic composition (δA) improved the accuracy in E/I ratio estimate over a simple precipitation equilibrium model, though a correction factor method provided nearly identical estimates of E/I ratio. The work demonstrates the feasibility of d-excess in the study of the water cycle for lakes in other regions of the world and provides recommendations on sampling strategies for accurate calculations of E/I ratio.