The hydrophobic fractionation of extracellular polymeric substances (EPS) extracted from anaerobic granular sludge was performed on the DAX-8 resin (two elution pH conditions, i.e., pH2 and pH5 were tested). The impact of seven different EPS extraction methods on EPS hydrophobicity features was assessed. The results showed that the extraction methods and bulk solution pH influenced dramatically the biochemical composition of the EPS, and in turn, the hydrophobicity determined. Besides, EPS extracting reagents i.e., formaldehyde, ethanol, sodium dodecyl sulfate (SDS), and Tween 20 not only introduced extra carbon content in the total organic carbon (TOC) measurement but also interacted with the DAX-8 resin. By comparing the apparent molecular weight (aMW) distribution of untreated and pH-adjusted EPS samples, more complete EPS aMW information was preserved at pH5. Thus, elution at pH5 was preferred in this study for the qualitative analysis of EPS hydrophobic features. The hydrophobic fraction of EPS retained by the resin at pH5 was ascribed to a wide aMW range, ranging from >440 to 0.3kDa. Within this range, EPS molecules ranging from 175 to 31kDa were mostly retained by the DAX-8 resin, which indicates that these EPS molecules are highly hydrophobic.