Abstract: Poly-β-hydroxybutyrate (PHB) in hydrogen-producing granular sludge was analyzed by ultraviolet (UV) spectrophotometry, which mainly included two steps: sludge pretreatment and UV determination. Sample of hydrogen-producing granular sludge was treated with NaClO and extracted with chloroform, 5 mL of H2SO4 solution was added and solution was heated in a water bath for 10 min, and its absorbance was measured at the wavelength of 235 nm. Linear relationship between values of absorbance and mass concentration of PHB was kept in the range within 4.0 mg·L-1 with detection limit (3s/k) of 0.023 mg·L-1. The proposed method was applied to the analysis of samples of hydrogen-producing granular sludge, giving values of recovery and RSD′s (n=6) in the ranges of 90.5%-108% and less than 2% respectively.