【Objective】It is to analyze the effects of three-stage seedling raising on rhizosphere microbial diversity and anti-stress enzyme activity of tobacco seedlings.【Method】Zhongyan 100 was chosen as the test variety, with conventional floating seedling raising as the control, and the three-stage seedling raising was set up. By monitoring the growth and development of tobacco seedlings in the quadruple sports seedling greenhouse, the effects of three-stage seedling raising method on seedling environment, microbial diversity, agronomic traits and anti-stress enzyme activities of tobacco seedlings were systematically analyzed. Concurrently, its effect on the microbial diversity of tobacco rhizosphere soil in the field was traced and analyzed.【Result】In the seedling stage, the three-stage seedling raising method improved the physical and chemical properties of the pool water and reduced the temperature difference and humidity level in the seedling greenhouse compared with the floating seedling raising control. The daily average temperature difference decreased from 19.23℃ to 17.95℃, and the daily average humidity decreased from 75.17%RH to 69.53%RH. At the seedling stage, the three-stage seedling raising increased the abundance of Brevundimonas and Lysinibacillus in the seedling pool water, which were 13.90 times and 4.66 times that of the floating seedling raising, respectively. The fresh weight, root length, number of lateral roots, CAT enzyme activity, SOD enzyme activity, POD enzyme activity, root activity and seedling rate of tobacco seedlings increased by 34.33%, 34.37%, 36.41%, 31.91%, 25.34%, 58.07%, 29.08% and 5.91%, respectively, compared with the floating seedling raising control, while the MDA content decreased by 22.31%. At the same time, MDA content decreased by 22.31%. In the field period, the abundance of Ascomycota and Penicillium in tobacco rhizosphere soil increased, and a more favorable environment for tobacco growth was created.【Conclusion】The three-stage seedling raising method increased the abundance of beneficial bacteria in the rhizosphere of tobacco seedlings by improving the seedling raising environment, and significantly improved the agronomic traits and anti-stress enzyme activity of tobacco seedlings at the seedling stage, thus improving the quality of tobacco seedlings.