The feasibility of using Fe0/S2O82- conditioning (zero valence iron (ZVI)/persulfate (ps)) integrated with horizontal electro-dewatering (HED) to improve the dewaterability of activated sludge (AS) was evaluated. The removal and migration of free and bound water in the ZVI/ps-HED process were determined, along with the organic matter migration between the solid and liquid phases of AS biosolids. Results showed that the optimum conditioning dosages were determined as 0.35 g ZVI/g dry solids (DS) and 0.15 g ps/g DS on the basis of capillary suction time variation. The lowest final water content (WC) of 83.67% in AS occurred after the HED treatment at 40 V and 120 min as determined using the response surface methodology. Despite the disruption of AS flocs and microbial cells through SO4-· oxidation during the ZVI/ps conditioning, the particle size and mass fractal dimension of the AS flocs remained relatively stable as the zeta potential increased from -8.5 mV to -4.6 mV. The coagulation of Fe3+ from the oxidation of Fe2+ by ps contributed to the relatively stable condition, which favored the subsequent momentum of the electroosmotic flow. Moreover, the ZVI/ps conditioning decreased the bound water content (BWC, from 1.83 g/g DS to 1.38 g/g DS) and increased the free water content (FWC, from 60.27 g/g DS to 60.91 g/g DS), indicating a transformation from free water to bound water. After the ZVI/ps-HED process, the FWC and BWC were removed significantly with ratios of 96.32% and 79.78%, respectively. Along with water removal through the ZVI/ps-HED process, the organic matter in the supernatant/filtrate initially increased and further rose at the HED stage. The analysis of the extracellular polymeric substance (EPS) content showed that the proteins (PNs) and polysaccharides (PSs) at the liquid and solid phases decreased due to oxidative degradation. Furthermore, the ZVI/ps stage significantly reduced the PNs content of slime to 57.22%, the PSs content of EPS to 68.50%, and the PN-like substances in the slime and tightly bound EPS to 74.90% and 52.47%, respectively. In addition, the WC of AS correlated with the contents of PN and microbial by-product-like material in slime, as well as tryptophan in TB-EPS, and the low EPS content in AS indicated good dewaterability. Thus, ZVI/ps should be selected as a pretreatment prior to HED.