The characteristics of DOM chemical fractions in road runoff with different traffic density were analyzed using excitation emission matrix spectroscopy (EEMs) technology, UV-vis spectra, and FTIR spectroscopy. The results showed that hydrophobic organics were the dominant components in DOM of road runoff, and hydrophobic neutral organics was the most abundant fraction. The hydrophilic organics in DOM of road runoff were mainly composed of protein-like substances. Both protein-like substances and fulvic-like acid substances were found in the hydrophobic organic matters. A high degree of aromatization and a low degree of humification were detected in hydrophobic organics, whereas the characteristics of hydrophilic organics were opposite. Some functional groups such as isomerized carboxyl and unsaturated C=C bonds were featured in acidic organics, and ether and ester groups, phenol, and alcohol were detected in alkaline and neutral organics. The substance composition and characteristics of DOM chemical fractions in road runoff were not affected by the traffic density. Traffic density influenced the quantity of substances in each chemical fraction only. With traffic density increasing, the matter content and the aromatization degree of DOM chemical fractions in road runoff increased, whereas the degree of humification decreased.