An online, semi-continuous instrument to measure fine particle (PM<sub>2.5</sub>) reactive oxygen species (ROS) was developed based on the fluorescent probe 2'7'-dichlorofluorescin (DCFH). Parameters that influence probe response were first characterized to develop an optimal method for use in a field instrument. The online method used a mist chamber scrubber to collect total (gas plus particle) ROS components (ROS<sub>t</sub>) alternating with gas phase ROS (ROS<sub>g</sub>) by means of an inline filter. Particle phase ROS (ROS<sub>p</sub>) was determined by the difference between ROS<sub>t</sub> and ROS<sub>g</sub>. The instrument was deployed in urban Atlanta, Georgia, USA, and at a rural site during various seasons. Concentrations from the online instrument generally agreed well with those from an intensive filter measurement of ROS<sub>p</sub>. Concentrations of the ROS<sub>p</sub> measurements made with this instrument were lower than reported in other studies, often below the instrument's average limit of detection (0.15 nmol H<sub>2</sub>O<sub>2</sub> equivalents m<sup>−3</sup>). Mean ROS<sub>p</sub> concentrations were 0.26 nmol H<sub>2</sub>O<sub>2</sub> equivalents m<sup>−3</sup> at the Atlanta urban sites compared to 0.14 nmol H<sub>2</sub>O<sub>2</sub> equivalents m<sup>−3</sup> at the rural site.