A humic acid extracted from a chernozem soil was fractionated combining size exclusion chromatography and polyacrylamide electrophoresis (SEC-PAGE). Three fractions named A, B, and CþD, with different electrophoretic mobilities and molecular sizes (MS), were obtained and subsequently characterized by thermochemolysis and surface-enhanced Raman spectroscopy (SERS). The data confirmed that fraction A, with the higher MS, was more aliphatic than fractions B and CþD and, in turn, fractions with lower MS (B and CþD) denoted an enrichment in lignin residues. These structural features explain conformational changes when varying the pH in the humic fraction A and indicated that combination of the two techniques is a good approach for characterizing humic substances.