Abstract: Chilling stress, one of the most important limiting environmental factors, delays plants growth and development and reduces crops yield. The mechanisms of chilling stress is cell membrane damaged by chilling injury, which causes reactive oxygen species (ROS) overproduction such as superoxide (O₂^- ), hydroxyl radicals ( OH) and hydrogen peroxide (H₂O₂). ROS is a cytotoxic compound and a mediator for the induction of stress tolerance. To protect cellular and organelle membranes from ROS damage, plants have evolved various enzymatic and non-enzymatic defense mechanisms for detoxifying free radicals and reducing oxidative stress. The antioxidive enzymes include superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT), peroxidases (POD), etc. Non-enzymatic antioxidants include glutathione, ascorbate, etc. In agriculture, scientists have attempted to seek some effective external physical ways to help plants eliminate the overproduction of ROS and enhance plant tolerance to environmental stress. Our previous studies also showed that CO₂ laser irradiation could enhance chilling tolerance by increasing the activities of nitric oxide synthase (NOS), CAT, POD, SOD and the concentrations of NO and glutathione. However, little is known about effects of laser and exogenous nitric oxide on chilling tolerance of wheat seedling. To determine the effect of CO₂ laser and exogenous nitric oxide on chilling tolerance of wheat seedling, seeds were exposed to different treatments and some physiological and biochemical parameters measured in 7-day-old seedlings. The results showed that compared with chilling stress (CS), wheat seedling subjected to sodium nitroprusside (SNP) and then followed by chilling stress (SNP+CS), decreased the concentrations of H₂O₂ and O₂^- and at the same time increased the activities of SOD, APX, GR, CAT, POD and NOS, and also increased the concentrations of NO and protein and the lengths of roots and shoots. Moreover, CO₂ laser treatment followed by chilling stress (LR+CS) resulted in significant decrease in the concentrations of H₂O₂ and O₂^- , and increased activities of SOD, CAT, POD, APX GR, NOS and the concentrations of NO and protein and the lengths of roots and shoots. When wheat seedling was subjected to SNP and CO₂ laser followed by chilling stress (SNP+LR+CS), the concentrations of H₂O₂ and O₂^- was higher and the above mentioned enzymes and seedling growth lower than that of SNP+CS. The results showed that SNP+LR had identical positive effects on enhancing chilling tolerance in wheat seedling. However, the effect of SNP+LR was less than that of SNP treatment. The results suggested that SNP and laser enhanced wheat seedling tolerance and recommended for application alone in agriculture.