The objective of this study is to test whether hydrogen peroxide (H2O2) is involved in laser pretreatment induced drought tolerance in wheat seedlings due to its nature as a second messenger in stress responses. Plant is treated with 3 min CO2 laser pretreatment, laser pretreatment in combination with catalase (CAT), ascorbate (AsA) or diphenylene iodonium (DPI) and their effects on the lipid peroxidation, the activities of antioxidant enzymes and the concentration of photosynthesis pigment and seedlings growth and development were compared. The results show that 3 min laser pretreatment can enhance drought tolerance in wheat seedlings by decreasing the concentration of malondialdehyde (MDA) and increasing the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and the concentration of chlorophyll a, chlorophyll b, carotenoid and plant height, root length and root dry weight. But the promotive effect of laser pretreatment induced drought tolerance in wheat seedling is effectively reversed by the addition of CAT, AsA or DPI. The results suggest that H2O2 is involved in laser pretreatment induced drought tolerance in wheat seedlings and laser induced protective effect is likely related to NADPH oxidase-dependent H2O2 production.