Fermentation process of Escherichia coli DH5α harboring phbCAB operon was optimized to achieve highest PHB productivity. Neutral pH (7.0) is optimal for PHB production of E. coli that can be applied at industrial scale as neutral and basic environment (pH ≥ 7) permit bacteria growth. In this study, Plackett – Burman experiment design was used to evaluate the effects of various physical-chemical factors on PHB yield – in which Molasses amount, fermentation time and pH were proved to affect the most. This study also utilized response surface method (RSM) and central composite design (CCD) to achieve optimal value of molasses amount (120g/l), fermentation time (48h) and pH (7.0), which yieled 3.36g/l cell dried weight (CDW) and 0.87g/l PHB products. The design model was applied in 150ml aerated fermentor and 1.5l automatic fermentor, achieved 3,3g/l CDW with 0,86g/l (PHB) and 3,4g/l CDW with 0,9g/l PHB, respectively.