Background: Highland barley has good resistance to low temperature and is the most commonly used pasture and greenery in high altitude area. Beyond that, Artemisinin is an allelochemical in the common weed Artemisia annua and remains in the soil, which affects the growth and development of highland barley. Most studies on its resistance are under single stress, while plants are often affected by many factors in reality. In this paper, highland barley was used to study the effects of combined stress of simulated freeze-thaw and artemisinin with different concentrations (10mg/L, 30mg/L, 50mg/L) on antioxidant enzyme activities, photosynthesis and osmotic regulation substance concentrations.The response characteristics of physiological characteristics under combined stress can study the damage mechanism of artemisinin on highland barley and provide a theoretical basis for reducing the damage caused by freeze-thaw to the growth of barley.Results: The results showed that under freeze-thaw stress, malondialdehyde(MDA) and soluble sugar in leaves decreased significantly by 9.0-40.9% and 21.0-29.8%, respectively, catalase(CAT) continued to decrease during the freeze-thaw cycle, photosynthetic rate firstly decreased and then increased, superoxide dismutase(SOD) reached the minimum value of 1470.9U/mgprot at -5℃, and the soluble protein maintained a relatively stable state.As the concentration of artemisinin increased, MDA and soluble sugar firstly decreased and then increased, reaching the lowest concentration at 10mg/L. Soluble protein, CAT, SOD and photosynthetic rate first increased and then decreased.Conclusion: All measurements indicated that under the compound stress of freeze-thaw and artemisinin, highland barley seedlings showed high cold resistance, and low concentration of artemisinin exerted a certain promotion effect on the growth of highland barley seedlings. Besides artemisinin and freeze-thaw showed an antagonistic effect on highland barley seedlings, and low concentration of artemisinin can alleviate the damage caused by freeze-thaw to highland barley seedlings.