Chilling stress, one of the most important limiting environmental factors, delays plants growth and development and reduces crops yield. Chilling stress causes cell membrane damage, which triggers an excessive production of reactive oxygen species (ROS) such as superoxide (O₂^-), hydroxyl radicals (^•OH) and hydrogen peroxide (H₂O₂). ROS are cytotoxic compounds and mediators 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. Antioxidive enzymes include, among others, superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT) and peroxidases (POD). Non-enzymatic antioxidants include glutathione, ascorbate and others. 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 showed that CO₂ laser irradiation could enhance chilling tolerance by increasing the activity 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. Results showed that, compared to chilling stress (CS) alone, wheat seedling subjected to sodium nitroprusside (SNP) followed by chilling stress (SNP+CS) decreased the concentrations of H₂O₂ and O₂^- while activities of SOD, APX, GR, CAT, POD and NOS increased at the same time. Furthermore, (SNP+CS) also increased the concentrations of NO and protein and the lengths of roots and shoots. Similarly, CO₂ laser treatment followed by chilling stress (LR+CS) resulted in a significant decrease in the concentrations of H₂O₂ and O₂^-, and increased activities of SOD, APX, GR, CAT, POD and NOS, concentrations of NO and protein and root and shoot lengths. 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 but the activity of the aforementioned enzymes and seedlings’ growth were lower than in the SNP+CS treatment. The results showed that SNP+LR had identical positive effects on enhancing chilling tolerance in wheat seedling. However, the effects of SNP+LR treatment were lower than those of SNP treatment alone. The results suggests that SNP and CO₂ laser radiation enhance wheat seedling tolerance, thus recommended for application in agriculture.