This study proposed a two-step process optimization for enhanced cumulative isoprene production. This involves establishing an isoprene biosynthetic pathway in Escherichia coli BL21 DE-3 via up-regulation of native deoxy xylulose 5-phosphate (DXP) synthase (EcDXS), isopentenyl pyrophosphate isomerase (EcIPPI/EcIDI) and introducing isoprene synthase (PmIspS) from kudzu (Pueraria montana), followed by the process conditions (incubation temperature, time, and inducer concentration) optimization using the Box-Behnken design (BBD) approach. BBD showed the maximum cumulative isoprene production (160.15 mg L-1), productivity (11.18 mg L-1 h-1), and yield (7.69 mg gdcw-1) at the optimized process conditions (incubation temperature 27.32 ºC, incubation time 14.25 h, and inducer concentration 0.453 mM). The transgenic E. coli isoprene production, -productivity, and -yield were 1.52-, 1.46-, and 1.24-fold higher than the unoptimized condition (incubation temperature 30 ºC, incubation time 16 h, and inducer concentration 0.10 mM). This work demonstrates that fine tuning of MEP pathway in addition with process conditions optimization is an efficient strategy for improving isoprene production from engineered E. coli.