For pollution monitoring, we developed a new combined fractionation technique (CFT) to characterize dissolved organic matter (DOM) in natural water by combining resin adsorption (RA) and ultrafiltration (UF) with 3D-fluorescence measurement. We tested the new technique on 4 polluted and 4 unpolluted samples. The 3D-fluorescence characteristics of size sub-components in the hydrophobic acid (HPOA) fraction could distinguish unpolluted from polluted DOM. The unpolluted HPOA fraction was composed of a single dominant size component - peak A (fulvic-like, around Ex240/Em410 nm) material with relatively large molecular weight (MW) (> 10 kDa). In comparison, the HPOA in polluted DOM contained another predominant size component with lower MW (< 5 kDa) - peak T material (tryptophan-like protein, around Ex230/Em340 nm). The fluorescence of peak T material with lower MW (< 5 kDa) in HPOA would be a good indicator of pollution or deterioration of source water quality. The application of this new CFT could yield more detailed and scientific information on the size and chemical character of the fluorophores in DOM sub-fractions.