Non-structural carbohydrates in plant organs can mirror the plant overall carbon supply status and balance and can also provide evidence for their health evaluation in the ecosystem. Non-structural carbohydrates in Kobresia pygmaea, one dominant herbaceous species on the Qinghai-Tibetan Plateau, were measured to investigate altitudinal variation in non-structural carbohydrate accumulations, as well as engineering disturbance on the stability of the alpine grassland ecosystem. An increasing trend with elevation in total soluble sugars, fructose, and sucrose was detected in the K. pygmaea growing in both undisturbed and disturbed sites. However, there were higher amounts and a more distinctly altitudinal trend of non-structural carbohydrates with a minor fluctuation in undisturbed sites compared to disturbed sites. In addition, the altitudinal trend of sucrose is similar to that in sucrose phosphate synthase and sucrose synthase activities, while it is opposite to that in neutral invertase and soluble acid invertase activities, suggesting that the sucrose accumulation was primarily related to its synthesis. These results revealed that human disturbance resulted in a reduced carbon supply and altered the balance of carbohydrate utilization in plants on the Qinghai-Tibetan Plateau.