Abstract: The application of plant ash is aimed at improving the soil microbial community structure, reducing the soil-borne diseases caused by the degeneration of the soil itself and better maintaining the balance of the soil ecosystem. A field experiment was conducted to explore the effects of applying plant ash on rhizosphere soil microbial quantity and functional diversity of flue-cured tobacco using plate culture method and Biolog technique. The results showed that the application of plant ash all significantly increased the amounts of bacteria, actinomycetes and the total microbes in rhizosphere of flue-cured tobacco, while the number of fungi decreased slightly at 35 d, 55 d and 75 d after transplanting respectively, the difference was not significant. Meanwhile, the utilization of carbon sources (AWCD) and six types of carbon sources in rhizosphere microbial communities of flue-cured tobacco was also significantly higher than that of the control during the periods mentioned above. The diversity indices (Shannon index, Simpson index, Evenness index and Richness index) of the rhizosphere soil microbial treated by plant ash were increased in all periods, and the difference was significant at 35 days after transplanting. Principal component analysis also suggested that the difference between the plant ash treatment and the control was obvious in principal component coordinate system with the plant ash treatment distributing in positive direction of PC1 while the control distributing in negative direction, indicating significant differences in soil microbial functional diversity. The above results showed that applying plant ash significantly improved the microbial community structure and functional diversity in rhizosphere soil of tobacco plants.