Abstract: T8 is a new flue-cured tobacco mutant line, which was selected from the Zhongyan100 EMS mutant library to have obvious fresh-sweet fragrance at bud stage. In order to comprehensively understand the formation of flavor at the molecular and genetic levels, we analyzed T8 and Zhongyan100 fresh tobacco leaves using the headspace-arrow solid phase micro-extraction and gas chromatography mass spectrometry (HS-SPME Arrow-GC/MS) to detect volatile organic compounds (VOCs), and the results were analyzed in combination with with RNA-seq data. The results showed that:(1) There are 26 kinds of VOCs in T8, among which 18 kinds of terpenes were found, and the relative content of terpenoids was 71.74%, which was 2.34 times of that of Zhongyan100. (2) The relative content of T8 linalool was 52.12%, which was 3.40 times of Zhongyan100, and it was the main component of fresh-sweet flavor. (3) In the MVA pathways of T8 young leaves at bud stage, 9 key rate-limiting enzyme genes and 12 TPS genes were up-regulated, which contributed in increasing the release of terpenoids. In summary, the synergistic up-regulation of several key genes in the MVA pathway contributes to the increase of overall release amount of terpenoids, and linalool with sweet flavor accounts for the highest proportion, which may be the main contributing factor to fresh-sweet flavor.