In order to reveal the function of the sterol C24 methyltransferase 2 gene (DoSMT2) in the sterol metabolism process of Dendrobium officinale, we firstly transformed the DoSMT2 gene into tobacco (Nicotiana tabacum) by Agrobacterium tumefaciens mediated method. qRTPCR was then applied to detected the DoSMT2 gene expression in transgenic tobacco leaves, and the contents of brassinosterol and sitosterol were analyzed by GCMS. The results showed that: (1) a 1 119bp openreadingframe (ORF) of DoSMT2 gene was successfully obtained, thereafter the sense plant expression vector pCXSNDoSMT2 was successfully constructed too. Four positive transgenic tobacco plants were obtained and identified by tobacco leaf disc transformation through A. tumefaciens mediated method. (2) Southern blot results showed that there was a hybridization signal band in all the four transgenic tobacco plants, while it was not found in nontransgenic tobacco plants, which indicated that the foreign gene DoSMT2 was integrated into the genome of the transgenic tobacco plants in a singlecopy mode. (3) The DoSMT2 gene expression was not detected in nontransgenic tobacco, while the expression of DoSMT2 gene could be detected in all four transgenic tobacco plants by qRTPCR, and the difference of expression level was significant. The expression level of each line was P3 > P1 > P2(P4), in turn. (4) The GCMS analysis results showed that the content of campesterol in transgenic tobacco leaves was significantly lower than that in nontransgenic tobacco leaves, while the content of sitosterol in transgenic tobacco leaves was significantly higher than that in nontransgenic tobacco leaves. This study indicated that the DoSMT2 protein had the catalytic activity in the transformation from 24methylene lophenol to 24ethylidene lophenol.