Abstract: Low content of neutral aroma component (NAC), lack of aroma and flavor and the decline in commodity characteristics caused by long-time continuous mono-cropping and irrational fertilization have been the serious problems in flue-cured tobacco production in east of Gansu Province. A field experiment was carried out in Zhengning County in Gansu Province in 2009 to study the effects of bio-organic fertilizer (BIO) application on NAC content in flue-cured tobacco and its mechanisms. BIO significantly increased the total content of NAC in tobacco leaves by 26.79%-134.33% compared with CK. The total content of NAC at different position of tobacco leaves had dramatically different reaction to BIO, namely, the biggest increase was observed in the upper leaves, and the total content of NAC in upper leaves, middle leaves and bottom leaves were increased by 134.33%, 81.03% and 26.79% respectively compared with CK. BIO application also changed the orders of the total content of NAC in tobacco leaves at different positions, the order of upper leaves＞bottom leaves＞middle leaves in CK treatment, were turned into upper leaves＞middle leaves＞bottom leaves in BIO treatment, which is favorable to the output value of tobacco production. The main NAC in tobacco leaves in Zhengning is neophytadiene, accounting for 79.67%-89.43% of the total amount. The neophytadiene content in upper, middle and bottom leaves of BIO treatment were increased significantly by 157.33%, 88.15% and 31.17% respectively compared with CK. BIO improved the nitrogen absorption by tobacco, which not only increased the chlorophyll content in tobacco leaves, the precursor of neophytadiene, but postponed the summit of chlorophyll content by about 15 days as well which could extend the mature period. Activities of invertase and nitrate reductase at round top stage in BIO treatment were increased by 30.02% and 13.42% compared with CK, which contributed to the dry matter accumulation, nitrogen uptake, as well as to the synthesis of NAC and chlorophyll.