Tomato seedlings were used as experimental materials and treated with 1.0, 2.0, 3.0, and 4.0 mg/L ozone water irrigation and 0.2, 0.4, 0.6, and 0.8 mg/L ozone water spray treatments. Indexes including the malondialdehyde (MDA) content, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), activities, soil and plant analysis development (SPAD) value, and nitrogen content of leaves were measured. Furthermore, the expression of antioxidant enzyme, chlorophyll synthesis and nitrogen absorption genes was analyzed after optimal ozone water treatment. The results showed that the activities of antioxidant enzymes in tomato leaves were significantly increased, and the MDA content in tomato leaves was significantly reduced by ozone water irrigation and spray treatment, which indicated that ozone water treatment can significantly improve the stress tolerance of tomato seedlings. Ozone water irrigation and spraying could also significantly increase the leaf SPAD value and nitrogen content of tomato seedlings, and the optimal concentrations of ozone water irrigation and spraying were 3.0 mg/L and 0.6 mg/L, respectively. The effect of ozone water irrigation on improving the physiological characteristics of tomato seedlings was better than that of spraying. After treatment with the optimal concentration of ozone water, the relative expression of antioxidant enzyme, chlorophyll synthesis, and nitrogen absorption genes was significantly increased, and the maximum expression level was reached at 12 h. In addition, ozone water irrigation could promote the expression of genes more than ozone water spraying, which was consistent with the improvements in the physiological characteristics of the tomato seedlings.