Isoprenoids, or terpenoids, have wide applications in food, feed, pharmaceutical, and cosmetic industries. Nerolidol, an acyclic C15 isoprenoid, is widely used in cosmetics, food, and personal care products. Current supply of nerolidol is mainly from plant extraction that is inefficient, costly, and of inconsistent quality. Here, we screened various nerolidol synthases from bacteria, fungi, and plants and found that the strawberry nerolidol synthase was most active in Escherichia coli. Through systematic optimization of the biosynthetic pathways, carbon sources, inducer, and genome editing, we constructed a series of deletion strains (single mutants ΔldhA, ΔpoxB, ΔpflB, and ΔtnaA; double mutants ΔadhE-ΔldhA; and triple mutants and beyond ΔadhE-ΔldhA-ΔpflB and ΔadhE-ΔldhA-ΔackA-pta) that produced high yields of 100% trans-nerolidol. In flasks, the highest nerolidol titers were 1.8 and 3.3 g/L in glucose-only and glucose–lactose–glycerol media, respectively. The highest yield reached 26.2% (g/g), >90% of the theoretic yield. In two-phase extractive fed-batch fermentation, our strain produced ∼16 g/L nerolidol within 4 days with about 9% carbon yield (g/g). In a single-phase fed-batch fermentation, the strain produced >6.8 g/L nerolidol in 3 days. To the best of our knowledge, our titers and productivity are the highest in the literature, paving the way for future commercialization and inspiring biosynthesis of other isoprenoids.