Ambient particulate matter (PM) has been demonstrated to cause adverse health outcomes, which are associated with oxidative stress derived from PM-mediated reactive oxygen species (ROS) generation. Humic-like substances (HULIS) are an abundant class of water soluble organic compounds (WSOC) from atmospheric aerosol particles and have been shown to mediate in ROS generation. In this study, a cell-free dithiothreitol (DTT) assay was used to measure the production of ROS mediated by HULIS isolated from different aerosol samples including ambient aerosols collected at rural, suburban and urban locations in the Pearl River Delta, China, as well as biomass burning smoke particles emitted from rice straw and sugarcane leaves burning. The result shows that biomass burning derived HULIS possess higher ROS generation potential than HULIS from typical urban aerosols. The DTT consumption rate of WSOC of ambient aerosols correlates well with the concentration of HULIS. HULIS was found to be the major redox active constituent of the water-extractable organic fraction in PM. In addition, nitrogen compounds that are components of HULIS, such as pyrrole, pyridine, pyrimidine, pyrazine, imidazole, n-methylimidazole and 3-methoxypyridine, have individually shown lack of capability in catalyzing the generation of ROS in the DTT assay on their own, but a synergistic effect is observed in that these compounds enhance the ROS generation of quinones known to be ROS active.