As an energy-efficient and eco-friendly sludge treatment process, two-stage anaerobic digestion (AD) is widely employed to recovery biomass energy from waste sludge. However, the effect of primary and secondary sludge for two-stage AD was not clear. In this study, two-stage AD of mixed sludge in different volume ratio was investigated. The maximum cumulative H2 yield (100.5 ml) and CH4 yield (2643.6 ml) were obtained in volume ratio of 1:3 (primary sludge: secondary sludge). In two-phase AD, mixed sludge could induce positive effect on both organics releasing in extracellular polymeric substances (EPS) and the utilization of volatile fatty acids (VFAs). By investigating the compositional characteristics of dissolved organic matters (DOM) through excitation-emission matrix (EEM) coupling with fluorescence regional integration (FRI), it revealed more degradable substances utilization in mixture of sludge. Results from this work suggest that two-phase AD with mixed sludge is efficient for renewable energy recovery.