To explore the physiological response of tobacco plant infected by Phytophthora to Trichoderma harzianum application sequence, the pot experiments were carried out with different treatments including inoculated with Phytophthora spore suspension (T1), inoculated with T. harzianum spore suspension (T2), inoculated with Phytophthora spore suspension first and then inoculated with T. harzianum spore suspension (T3), inoculated with T.harzianum spore suspension first and then inoculated with Phytophthora spore suspension (T4), inoculated with Phytophthora spore suspension and T. harzianum spore suspension at the same time (T5), and clear water (CK) as controls. The effects of T. harzianum application sequence on biological characteristics, physiological characteristics and induced resistance of tobacco infected by Phytophthora were analyzed. The results showed that, in the growth of tobacco plants, the agronomic characters of tobacco plants with T4 treatment increased the most, and the dry and fresh weight of underground parts increased by 103.73% and 8.30% compared with CK. In the physiological characteristics of tobacco plants, T. harzianum could reduce the inhibition of Phytophthora on photosynthetic pigment accumulation and root activity of tobacco plants, and significantly increase both of them, and it could also strengthen the activities of enzymes related to carbon and nitrogen metabolism in tobacco plants. After transplanting 21 d, the activities of nitrate reductase and glutamine synthetase with T4 treatment increased by 13.14% and 6.40%, respectively. In the control effect, T4 treatment had the lowest incidence and disease index, which were 39.39% and 13.89%, and the highest antioxidant enzyme activity. In conclusion, there were significant differences in the physiological response of tobacco plants to the inoculation sequence of T. harzianum. In production, T. harzianum should be inoculated in advance to promote the growth and development of tobacco and inhibit the incidence of black shank disease. Above results laid a theoretical foundation for the regulation of tobacco growth and development and the comprehensive prevention and control of black shank.