Polyhydroxybutyrate (PHB) was produced by activated sludge in a two-stage process. In the first stage, the sludge with high storage potential was enriched in a sequencing batch reactor (SBR) operated by a dynamic feeding regime. The biosynthesis of PHB was sequentially accomplished in a batch reactor in the second stage. This article focused on the optimization of PHB content by evaluating the effects of three critical factors: Dissolved oxygen, pH and food-to-microorganism (F/M) ratio in the batch reactor. The results showed that the rate of substrate uptake, as well as the yield and content of PHB increased with the increase of dissolved oxygen concentration. The control of pH was not necessary under the weak alkaline condition for improvement of PHB content, except for the case in which the pH value was out of rang of 7.0 to 10.0. The enhanced F/M ratio favored PHB accumulation in the range of 1.0 to 4.5 C-mmol/C-mmol and the highest PHB content, 64%, was achieved at 4.5 C-mmol/C-mmol. When F/M ratio was enhanced to 6.0 C-mmol/C-mmol, the phenomenon of utilizing simultaneously intracellular PHB and external substrate by sludge was observed, which resulted in a sharp decrease of PHB content. The microscopic observation clearly confirmed that dynamic feeding regime was effective for selecting and enriching the sludge with a high storage potential.polyhydroxybutyrate, activated sludge, dynamic feeding regime, optimum operating conditions Conventional plastics have faced some severe problems, because of their persistency in the environment and non-biodegradability. Therefore, the biodegradable plastic has potentials in applications. Polyhydroxyalkanoates (PHAs) are the polyester of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds, and have similar mechanical properties to conventional plastics like polypropylene or polyethylene. Additionally, PHAs have the attractive features of being completely biodegradable, biocompatible, and produced from renewable sources. Polyhydroxybutyrate (PHB) is the most common and best characterized PHAs. For the economical production of PHAs, various bacterial strains, either wild-type or recombinant, and new fermentation strategies were developed with high content and productivity of PHAs [1,2]