To investigate the mechanism by which laser irradiation enhances the chilling tolerance of wheat seedlings, seeds were exposed to different treatments, and biochemical parameters were measured. Compared with the control group, chilling stress (CS) led to an increase in the concentrations of malondialdehyde (MDA) and H₂O₂, and decreases in the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT), peroxidase (POD), and nitric oxide synthase (NOS), and the concentrations of nitric oxide (NO) and protein. Treatment with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), sodium tungstate (ST), and NG-nitro-L-arginine methyl ester (L-NAME) followed by CS resulted in further increases in the concentrations of MDA and H₂O₂ and further decreases in the other parameters. However, treatment with PTIO, ST, and L-NAME followed by laser irradiation had the opposite effects on these parameters. When the seeds were treated with PTIO, ST, and L-NAME followed by laser and CS, the concentrations of MDA and H₂O₂ were significantly lower and the other parameters were higher than in the PTIO, ST, and L-NAME plus CS groups. These results suggest that CO₂ laser irradiation enhances the chilling tolerance of wheat seedlings by stimulating endogenous NO synthesis.