In order to study the biosynthesis of sesquiterpene, this text analyzed the sesquiterpene synthetases gene(AcTPS2) through genome analysis of Antrodia camphorata, and obtained the full length cDNA with RTPCR, carried on bioinformatics and mRNA analysis. The results showed that AcTPS2 cDNA has 1 086 bp; Blast analysis revealed that AcTPS2 included abundant asparagic acid motif DXXXD that is specific motif of sesquiterpene synthetase, and terpene synthetase special conserved sequence RRDTSGLDL; phylogenetic trees showed AcTPS2 and other fungus sesquiterpene synthetase amino acid sequence clustered in one clade; mRNA analysis showed manitol as carbon source, casein peptone as nitrogen source could promote AcTPS2 effectively, and the results could supply some basis in sesquiterpene biosynthesis.