Biodiesel, or fatty acid ethyl ester (FAEE), is an environmentally safe, next-generation biofuel. Conventionally, FAEE is produced by the conversion of oil/fats, obtained from plants, animals, and microorganisms, by transesterification. Recently, metabolic engineering of bacteria for ready-to-use biodiesel was developed. In Escherichia coli, it is produced by fatty acyl-carrier proteins and ethanol, with the help of thioesterase (TesB) and wax synthase (WS) enzymes. One of the foremost barriers in microbial FAEE production is the feedback inhibition of the fatty acid (FA) operon (fabHDG). Here, we studied the effect of biodiesel biosynthesis in E. coli with an engineered fabHDG operon. With a basic FAEE producing BD1 strain harboring tes and ws genes, biodiesel of 32 mg/L were produced. Optimal FAEE biosynthesis was achieved in the BD2 strain that carries an overexpressed operon (fabH, fabD, and fabG genes) and achieved up to 1291 mg/L of biodiesel, a 40-fold rise compared to the BD1 strain. The composition of FAEE obtained from the BD2 strain was 65% (C10:C2, decanoic acid ethyl ester) and 35% (C12:C2, dodecanoic acid ethyl ester). Our findings indicate that overexpression of the native FA operon, along with FAEE biosynthesis enzymes, improved biodiesel biosynthesis in E. coli.