Z,Z-farnesol (Z,Z-FOH), the all-cisisomer of farnesol, holds enormous potential for application in cosmetics, daily chemicals, and pharmaceuticals. In this study, we aimed to metabolically engineerEscherichia colito produce Z,Z-FOH. First, we tested five Z,Z-farnesyl diphosphate (Z,Z-FPP) synthases that catalyze neryl diphosphate to form Z,Z-FPP inE. coli. Furthermore, we screened thirteen phosphatases that could facilitate the dephosphorylation of Z,Z-FPP to produce Z,Z-FOH. Finally, through site-directed mutagenesis ofcis-prenyltransferase, the optimal mutant strain was able to produce 572.13 mg/L Z,Z-FOH by batch fermentation in a shake flask. This achievement represents the highest reported titer of Z,Z-FOH in microbes to date. Notably, this is the first report on the de novo biosynthesis of Z,Z-FOH inE. coli. This work represents a promising step toward developing syntheticE. colicell factories for the de novo biosynthesis of Z,Z-FOH and othercis-configuration terpenoids.