Soil microbial community can indicate soil quality change and is regarded as the early warning and the sensitive indicator. In order to study the effects of green manure application combined with chemical fertilizers on soil microbial community structure of tobacco-planting soils, the phospholipid fatty acids (PLFAs) were determined in 3a located experiment. The results showed that fertilization increased the kinds and amounts of soil PLFAs. The bacterial PLFAs, arbuscular mycorrhizal fungi PLFAs and total soil microbial PLFAs were enhanced distinctly when the amount of chemical fertilizers used more than 85% of conventional dosage with burying 15 000 kg/hm2 green manure. The negative effect on soil microbial community structure was appeared when the amount of chemical fertilizers was used less than 70% of conventional dosage with burying 15 000 kg/hm2 green manure. Principal component analysis and cluster analysis indicated that soil microbial community structure changed due to the ratio of green manure and chemical fertilizers. Correlation analysis showed the high agreement between chloroform-fumigation extraction and PLFA methods, available potassium and phosphorus were significantly positively correlated with the G-PLFAs, but not correlated with fungi PLFAs.