The major phytochemicals in the roots of Cudrania tricuspidata are prenylated xanthones, exhibiting significant structural diversity and bioactive properties, such as anti-inflammatory, antioxidative, and antitumor effects. The biosynthetic pathways of these compounds have not yet been resolved, limiting their production through synthetic biology. In this study, benzoyl-coenzyme A (CoA) ligase (BZL), benzophenone synthase (BPS), and benzophenone 3'-hydroxylase (B3'H) transcripts involved in the biosynthesis of xanthone were cloned and characterized from C. tricuspidata. The results showed that C. tricuspidata BZL (CtBZL) catalyzed the formation of benzoyl-CoA from benzoate and C. tricuspidata BPS (CtBPS) catalyzed the condensation of benzoyl-CoA and three molecules of malonyl-CoA to form 2,4,6-trihydroxybenzophenone (2,4,6-triHB) via Claisen type cyclization. Then, C. tricuspidata B3'H (CtB3'H) hydroxylated 2,4,6-triHB to produce 2,3',4,6-tetrahydroxybenzophenone (2,3',4,6-tetraHB), which was a key precursor of xanthone derivatives. In addition, the functions of the enzymes were further explored by the heterologous expression of CtBZL, CtBPS, and CtB3'H in Saccharomyces cerevisiae and Nicotiana benthamiana. Also, a preliminary heterologous synthesis system of 2,3',4,6-tetraHB was established. The present study provided the candidate genes and strategies for the heterologous production of xanthones using S. cerevisiae and N. benthamiana as hosts.