Inhibition by humic substances (HSs) on anaerobic digestion of excess activated sludge is one of the limitations for converting more organics into biogas. In order to counteract the inhibition from HSs, the present study was initiated to understand the dynamic changes (content and structure) of HSs during the anaerobic digestion of synthetic and real sludge. For the first time, the present work studied the dynamics of HSs separately in the liquid and solid phases. These observations on HSs conversions by potentiometric titration, UV–Vis, FTIR, and two-dimensional infrared spectra, confirmed that the dynamic changes of HA and FA compositions were caused by losing aliphatic moieties and enriching aromatic moieties in the structural compositions. This changes increased the humification degree, aromaticity, and the amounts of oxygen-containing functional groups. Based on the observations, strategies to alleviate the inhibition effect were discussed.