The enzyme-accelerated biodegradation of total petroleum hydrocarbons (TPH) was investigated in a sequencing continuous-inflow reactor (SCR) at different operational parameters of H2O2/TPH ratio, initial TPH concentration and hydraulic retention time (HRT). The optimum H2O2/TPH mass ratio was determined to be 0.35 at which the complete TPH removal of inlet TPH concentrations up to 4 g/L at HRT of 24 h, corresponding to the loading rate of 41cg TPH/m(3).d, was attained. The average COD removal efficiency at this loading rate was 96.7%. With increasing the inlet TPH concentration from 1 to 2.5 g/L, the biomass bacterial activity as dehydrogenase activity (DHA) increased from 7.5 to 27.1 mu g TF/g(biomass) d and remained almost unchanged with further increase of TPH concentration. The peroxidase activity (PA) remained high between 382 and 410 U/g(biomass). In addition, the complete removal of 1 TPH (88.7% COD removal) was observed at HRT of as small as 4 h (corresponding to the loading rate of 6 kg TPH/m3.d) under optimum H202/TPH mass ratio. With the decrease of HRT from 24 h to 4 h at the constant TPH concentration of 1 g/L the value of DHA remained between 24.4 and 28.4 mu g TF/g(biomass).d while the PA value increased from 287.9 to 394.4 U/g(biomass). Total production of biosurfactants was 131 mg/L (38 mg/L rhamnolipid and 93 mg/L surfactin) when the SCR was operated at TPH loading rate of 6 kg/m(3).d. Finally, the enhanced enzymatic biodegradation of TPH by using diverse microbial consortia capable of in-situ production of peroxidase and biosurfactant generation in the SCR is a very efficient and promising technique for accelerated biodegradation and COD removal of petroleum hydrocarbons. 2016 Elsevier B.V. All rights reserved.