This study was carried out to comparatively evaluate the role of extracellular polymeric substances (EPS) in biosorption of Ni(II) onto aerobic/anaerobic granular sludge (AGS and AnGS). Batch sorption experiments data suggested that Ni(II) sorption onto AGS and AnGS were better fitted to Langmuir and Freundlich model, respectively. The contribution of EPS in the sorption of AnGS was higher than that of AGS. Interaction between EPS and Ni(II) was investigated by a combined three-dimensional excitation-emission matrix (3D-EEM), synchronous fluorescence spectra, two-dimensional correlation spectroscopy (2D-COS) and fourier transform infrared spectroscopy (FTIR). 3D-EEM showed that protein (PN)-like substances were the main peaks in EPS from AGS and AnGS. The quenched types of AGS and AnGS in the presence of Ni(II) belonged to dynamic and static quenching, respectively. Additionally, 2D-COS demonstrated that fluorescence change took place sequentially followed to humic-like region > PN-like region in AGS and fulvic-like fraction > PN-like and humic-like fractions in AnGS. According to FTIR spectra, hydroxyl group and amino group in both EPS were the key functional groups for Ni(II) sorption. (C) 2016 Elsevier Inc. All rights reserved.