Pressurized vertical electro-osmotic dewatering (PVEOD) is an emerging mechanical technology for deep dewatering of wastewater sludge. In this study, the influence of main operation variables including sludge dosage, voltage, mechanical pressure on electro-osmotic dewatering was investigated. Response surface method (RSM) was applied to evaluate and optimize the interactive effects of the three major oper-ating variables. The results indicated the optimum conditions to be 2.70 MPa of mechanical pressure, 18.33 V of voltage and 0.0169 kWh/kg water of energy consumption. Layered distribution study was con-ducted between anode side and cathode side to investigate the vertical changes of moisture content, zeta potential, three-dimensional excitation emission matrix (3D-EEM) spectra of extracellular polymeric sub-stances, the concentration of proteins and polysaccharides. The morphology changes of sludge observed by microscope images unveiled the possible mechanisms of enhancing the sludge dewaterability in the PVEOD process. The electrochemical at electrodes and electro-osmosis are important for interpreting the dryer sludge at anode layer. (C) 2020 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.