(R)-hydroxyalkanoic acids (R-HAs) are valuable building blocks for the synthesis of fine chemicals and biopolymers because of the chiral center and the two active functional groups. Hydroxyalkanoic acids fermentation can revolutionize the polyhydroxyalkanoic acids (PHA) production by increasing efficiency and enhancing product utility. Modifying the fermentation conditions that promotes the in vivo depolymerization and secretion to fermentation broth in wild type bacteria is a novel and promising approach to produce R-HAs. Wood extract hydrolysate (WEH) was found to be a suitable substrate for R-3-hydroxybutyric acid (R-3-HB) production by Burkholderia cepacia. Using Paulownia elongate WEH as a feedstock, the R-3-HB concentration in fermentation broth reached as high as 14.2 g/L after 3 days of batch fermentation and the highest concentration of 16.8 g/L was obtained at day 9. Further investigation indicated that the composition of culture medium contributed to the enhanced R-3-HB production.