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Abstract Tryptophol (IET) is a metabolite derived from L-tryptophan that can be isolated from plants, bacteria, and fungi and has a wide range of biological activities in living systems. Despite the fact that IET biosynthesis pathways exist naturally in living organisms, industrial-scale production of IET and its derivatives is solely based on environmentally unfriendly chemical conversion. With diminishing petroleum reserves and a significant increase in global demand in all major commercial segments, it becomes essential to develop new technologies to produce chemicals from renewable resources and under mild conditions, such as microbial fermentation. Here we characterized and engineered the less-studied L-tryptophan pathway and IET biosynthesis in the baker’s yeast Saccharomyces cerevisiae, with the goal of investigating microbial fermentation as an alternative/green strategy to produce IET. In detail, we divided the aromatic amino acids (AAAs) metabolism related to IET synthesis into the shikimate pathway, the L-tryptophan pathway, the competing L-tyrosine/L-phenylalanine pathways, and the Ehrlich pathway based on a modular engineering concept. Through stepwise engineering of these modules, we obtained a yeast mutant capable of producing IET up to 1.04 g/L through fed-batch fermentation, a ~ 650-fold improvement over the wild-type strain. Besides, our engineering process also revealed many insights about the regulation of AAAs metabolism in S. cerevisiae. Finally, during our engineering process, we also discovered yeast mutants that accumulate anthranilate and L-tryptophan, both of which are precursors of various valuable secondary metabolites from fungi and plants. These strains could be developed to the chassis for natural product biosynthesis upon introducing heterologous pathways.