Abstract: In this study, a by-product of sugar industry was used as raw material, and levulinic acid (LA) was prepared by acid hydrolysis from molasses, and hydrolyysate fermentation with LA-rich produce biodegradable plastic polyhydroxybutyrate (PHB). In this paper, single factor and response surface method were used to optimize the hydrolysis conditions of molassic acid. The optimal reaction conditions were worked out and were as follows: liquid-solid ratio 1.24 mL/g, reaction temperature 146 ℃, reaction time 1 h. Under this condition, the highest yield of LA reached 44.09%, significantly better than LA yield without optimization (17.39%), which was better than the yield of LA optimized by response surface method for the preparation of SUGARcane molasses catalyzed by sulfuric acid (30.11%). The biomass of Cupriavidus necator was 2.99 g/L after 48 h, and the PHB content of Cupriavidus necator reached 14.85% of cell dry weight. The results show that the optimized conditions of acid hydrolysis process could significantly improve the yield of LA from molasses. It is feasible to use the molasses hydrolysate rich in LA as a feedstock for PHB production.