In this study, we report the in vivo imaging of reactive oxygen species (ROS)-overproducing pro-inflammatory macrophages in atherosclerotic plaques using a fluorescent ROS nanosensor. We designed the nanosensor by chemically conjugating hyaluronic acid (HA, a targeting ligand for CD44 receptor), chlorin e6 [Ce6; a near-infrared fluorescent (NIRF) dye], and a thioketal (TK) linker (ROS-degradable linker). The self-assembled nanosensor emitted weak NIRF signals in normal physiological conditions, whereas it emitted strong NIRF signals under ROS-abundant conditions. The cytocompatible nanosensor showed higher intracellular internalization via receptor-mediated endocytosis, which enabled the visualization of intracellular ROS in pro-inflammatory macrophages. Moreover, we demonstrated that the nanosensor enabled the successful targeting and imaging of CD44- and ROS-overproducing pro-inflammatory macrophages in atherosclerotic plaques, as validated by confocal microscopy and immunohistological analyses. These data suggest that our ROS nanosensor is suitable for ROS imaging in pro-inflammatory macrophages in vitro and in vivo and will be a promising approach for imaging atherosclerotic tissues and evaluating the effects of antioxidants.