In order to investigate the physiological injury of barley caused by the low temperature after herbicides, tillering barley seedlings planted in plastic cups were pretreated in illumination incubator at 15℃ and 12 h-light per day for 7 days, and then subjected to herbicide treatment, prometryn (with the concentrations of 0, 0.15%, 0.30%, and 0.45%) or isoproturon (with the concentrations of 0, 0.30%, 0.60%, and 0.90%), and the SOD activity, the CAT activity, the POD activity, the MDA content, proline content, soluble protein content, electrical conductivity, and the rate of O2-were determined and analyzed. The results showed that the low-temperature was the most important, and the treatment-time of low-temperature was another significant influencing factor on the physiological and biochemical indices of barley seedlings. However, all of the physiological and biochemical indices determined were not affected by the kinds of herbicides and herbicide concentrations, and the SOD was stable and should play the more prominent role on extracting of free radicals according to the stepwise regression and correlation. The herbicide concentrations should be increased in the future research for truly reflecting the effects of the herbicide concentration on the physiological and biochemical indices of barley seedlings. In order to investigate the physiological injury of barley caused by the low temperature after herbicides, tillering barley seedlings planted in plastic cups were pretreated in illumination incubator at 15 ° C and 12 h-light per day for 7 days, and then subjected to herbicide treatment, prometryn with the concentrations of 0, 0.15%, 0.30%, and 0.45% or isoproturon with the concentrations of 0, 0.30%, 0.60%, and 0.90%, and the SOD activity, the CAT activity, the POD activity , the MDA content, proline content, soluble protein content, electrical conductivity, and the rate of O2-were determined and analyzed. The results showed that the low-temperature was the most important, and the treatment-time of low-temperature was another significant influencing factor on the physiological and biochemical indices of barley seedlings. However, all of the physiological and biochemical indices determined were not affected by the kinds of herbicides and herbicide concentrations, and the SOD was stabl e and should play the more prominent role on extracting the free radicals according to the stepwise regression and correlation. The herbicide concentrations should be increased in the future research for truly reflecting the effects of the herbicide concentration on the physiological and biochemical indices of barley seedlings.