The pursuit of high-rate anaerobic reactor has powerfully promoted the studies of anaerobic granular sludge (AnGS) recently. In this work, Methylene Blue and Rhodamine B were used as hydrophilic/hydrophobic indicators to investigate the hydrophilicity/hydrophobicity of AnGS, and several analysis methods were applied to reveal the causes of difference in hydrophilicity/hydrophobicity between the surface and interior of AnGS. Results showed that hydrophilic/hydrophobic surface area ratios for intact and loose AnGS were 1.587 and 1.207, respectively, indicating that the surface was more hydrophilic than the interior. Such difference was ascribed to spatial distribution discrepancy of polysaccharides and proteins of extracellular polymeric substance. The different relative contents of polar groups like -OH, C@O, -NH2 and nonpolar groups like side chains of aromatic amino acids were suggested to be the primary causes that lead to the difference. The results are meaningful for the cultivation of high-activity AnGS and the development of anaerobic technology. (C) 2016 Elsevier Ltd. All rights reserved.