The wheat seeds are irridated by CO2 laser with power of 20.1 mW/mm2 for 3 min. When the wheat seeds grow about 12 days, the seedlings are treated with the 10% polyethyleneglycol 6000 (PEG6000) stress. And the effects of NO on the CO2 laser pretreatment inducing drought tolerance are studied through sodium nitroprusside and hemoglobin added with NO. The results show that laser pretreatment and NO donor sodium nitroprusside (SNP) could decrease the concentration of malondialdehyde (MDA) and increase the activities of superoxide dismutase (SOD), peroxidase(POD), catalase (CAT) and the concentration of chlorophyll a, chlorophyll b and root dry weight in the wheat seedlings with drought tolerance. But the promotive effect of laser pretreatment induced drought tolerance in wheat seedlings was not effectively reversed by the addition of hemoglobin (NO scavenger).