In this paper the production of poly (3-hydroxybutyrate/3-hydroxyvalerate) (PHBV) with activated sludge was investigated by using the fermentation liquid from primary sludge (PS) and waste activated sludge (WAS) as carbon source. First, the suitable concentration and ratio of acetic to propionic (acetic/propionic) for PHBV synthesis with desired hydroxyvalerate (HV) fraction was determined. Then, the conditions for producing fermentation liquid with the required acetic/propionic from the PS/WAS mixture were optimized. Finally, this optimized fermentation liquid was used as the carbon source for PHBV synthesis by the aerobic feeding and discharge process. The PHBV content in the sludge reached 65.5%, with 47.8% (mol-C based) of HV unit content, and the corresponding polyhydroxyalkanoates yield per litre of acclimated sludge was 1.44 g/L. This was the first optimal PHBV biosynthesis reported by using activated sludge as the microbe and sludge fermentation liquid as the carbon source. Polymerase chain reaction-based 454 pyrosequencing analysis revealed that Thauera strains were the predominant species in the PHBV biosynthesis system.