Poly-3-hydroxybutyrate (PHB) is mainly accumulated by Ralstonia eutropha under unbalanced growth conditions, which limits its production in batch or fed-batch modes. The continuous production of PHB was investigated in a two-stage continuous culture system. The first-stage produced cell mass giving the maximal cell dry weight of 27.1g l−1 at 0.21h−1 of dilution rate. High specific cell growth rate results in the decrease of PHB synthesis under glucose-limited and nitrogen-rich conditions in the first-stage. The second-stage produced PHB giving the maximal PHB concentration of 47.6g l−1 at 0.14 h−1 of dilution rate. Specific PHB synthetic rate reached highest value at low dilution rate under nitrogen-limited condition in the second-stage, and decreased with the increase of ammonium concentration in the culture. In the continuous culture system, the maximal PHB productivity could reach 1.43g l−1h−1 at a dilution rate of 0.12h−1, but with relatively low PHB content of 47.6%. Maximal yield of PHB on glucose could reach 0.36g g−1 glucose at 0.075h−1 of dilution rate with relatively high PHB productivity of 1.23g l−1h−1 and PHB content of 72.1%, respectively.