Diminishing petroleum reserves and the rapid accumulation of greenhouse gases lead to increasing interest in microbial biofuels. In this study, a heterologous farnesyl acetate biosynthesis pathway was constructed in Escherichia coli for the first time. Firstly, the AtoB, ERG13, tHMG1, ERG12, ERG8, MVD1, Idi, IspA and PgpB were expressed to accumulate farnesol in the E. coli cells. Then the alcohol acetyltransferase (ATF1) was heterologous overexpressed for the subsequent esterification farnesol to farnesyl acetate. The engineered strain DG 106 accumulated 128 +/- 10.5 mg/L of farnesyl acetate. Finally, the isopentenyl-diphosphate isomerase was further overexpressed, and the recombinant strain DG107 produced 201 +/- 11.7 mg/L of farnesyl acetate. This study shows the novel method for the biosynthesis of the advanced biofuel farnesyl acetate directly from glucose and highlight the enormous designing strategies for metabolic engineering of bioproducts.