In the Qinghai-Tibet Plateau, both the large daily temperature difference and soil salinization make plants susceptible to abiotic stresses such as freeze-thaw and salinity. Meanwhile, crops in this area could be subjected to the influence of artemisinin, an allelochemical exuded by Artemisia annua. In the context of freeze-thaw and salinity stresses, artemisinin was induced as an allelopathy stress factor to explore the physiological response of highland barley, including the relative electrical conductivity (RC), soluble protein (SP) content, malondialdehyde (MDA) content, antioxidant enzyme activity, and water use efficiency (WUE).There data suggested that artemisinin weakened the self-osmotic adjustment ability of seedlings, reducingthe SOD activity in scavenging efficiency of reactive oxygen species, then causing oxidative damage to cell membrane of seedlings, which significantly increases the content of RC and MDA. Artemisinin stress can reduce the WUE of seedlings and weaken the photosynthesis intensity of seedlings as well. In a word, salinitystress and artemisinin respectively showed a synergistic compound relationship with freeze-thaw stress.