Lignocellulose, derived from non-food crops, is a competitive renewable sugar source. In this study, it was pretreated to produce lignocellulose hydrolysate (reducing sugar), and then used as co-substrate to accumulate polyhydroxyalkanoate (PHA) with its fermented product volatile fatty acids (VFAs). The rule of PHA accumulation and structural characterization from single VFAs, lignocellulose hydrolysate and VFAs co-substrate two carbon sources were investigated. As a functional green material, it is expected to replace the traditional petroleum-based polymer. During the culture process, compared with single VFAs carbon source, higher PHA production of 3049 mg COD/L was achieved with the addition of co-substrate, especially the proportion of 3-hydroxyvalerate (HV) monomer reached 44.9 % at C/N ratio of 33. The average molecular weight of PHA was 1.16∼1.27 × 105 Da, with a polydispersity index of 2.08∼2.46. The synthesized PHA was thermal stable up to 285 °C, which was 14 °C higher than that of commercial poly (hydroxybutyrate-co-hydroxyvalerate) (PHBV). As a biopolymer film, it possessed high tensile strength (8.13 ± 0.607 MPa), Young's modulus (875.47 ± 26.21 MPa).