In this study, high pure humic acid (HA) extracted from lignite was fractionated into four fractions according to their molecular weights by the ultrafiltration method. The structural characteristics of various HA fractions were investigated by UV–Vis,FTIR, and 3D fluorescence spectra and 13 C/ 1 H-NMR tests. The experiment results showed that the HA fractions with the molecular weight >50 KDa are major, containing 70.89%. The active hydrogen proportion declined from 43.96% to 21.27% with the increase of HA molecular weight, meaning the decrease of the large HA molecules’ acidic group content. On the other hand, 80.93% of the aromatic nucleus content and 12.5% of the alkyl carbon (aliphatic structure) were observed in the large HA molecules that are much higher than those in the small HA molecules. The NMR and spectra studies indicated that the small HA molecules have more acidic groups such as hydroxy, carboxyl. Meanwhile, the large HA molecules have more aromatic and aliphatic structures. The above research indicated that the HA fractions with low molecular weight could provide more active sites for the interaction between HA and iron minerals. Moreover, the carbon chain and aromatic nucleus in the high molecular weight HA fractions could keep the space structure of HA stable. • High pure lignite humic acid was fractionated successfully by ultrafiltration. • The differences of composition and structure of various HA fractions were investigated. • The structure proportion of HA was quantized by integral and PARAFAC method. • The smaller HA molecular had more abundant oxygen-bearing groups. • The larger HA molecular had more aromatic structures.