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Dendrobium sinense, an endemic medicinal herb in Hainan Island, is rich in bibenzyls. However, the key rate-limited enzyme involved in bibenzyl biosynthesis has yet to be identified in D. sinense. In this study, to explore whether there is a significant difference between the D. sinense tissues, the total contents of bibenzyls were determined in roots, pseudobulbs, and leaves. The results indicated that roots had higher bibenzyl content than pseudobulbs and leaves. Subsequently, transcriptomic sequencings were conducted to excavate the genes encoding type III polyketide synthase (PKS). A total of six D. sinense PKS (DsPKS) genes were identified according to gene function annotation. Phylogenetic analysis classified the type III DsPKS genes into three groups. Importantly, the c93636.graph_c0 was clustered into bibenzyl synthase (BBS) group, named as D. sinense BBS (DsBBS). The expression analysis by FPKM and RT-qPCR indicated that DsBBS showed the highest expression levels in roots, displaying a positive correlation with bibenzyl contents in different tissues. Thus, the recombinant DsBBS-HisTag protein was constructed and expressed to study its catalytic activity. The molecular weight of the recombinant protein was verified to be approximately 45 kDa. Enzyme activity analysis indicated that the recombinant DsBBS-HisTag protein could use 4-coumaryol-CoA and malonyl-CoA as substrates for resveratrol production in vitro. The Vmax of the recombinant protein for the resveratrol production was 0.88 ± 0.07 pmol s−1 mg−1. These results improve our understanding with respect to the process of bibenzyl biosynthesis in D. sinense. [ABSTRACT FROM AUTHOR]