Macrophages play pivotal roles in the maintenance of tissue homeostasis. However, the reactivation of macrophages toward proinflammatory states correlates with a plethora of inflammatory diseases, including atherosclerosis, obesity, neurodegeneration, and bone marrow (BM) failure syndromes. The lack of methods to reveal macrophage phenotype and function in vivo impedes the translational research of these diseases. Here, we found that proinflammatory macrophages accumulate intracellular lipid droplets (LDs) relative to resting or noninflammatory macrophages both in vitro and in vivo, indicating that LD accumulation serves as a structural biomarker for macrophage phenotyping. To realize the staining and imaging of macrophage LDs in vivo, we developed a fluorescent fatty acid analog-loaded poly(lactic-co-glycolic acid) nanoparticle to label macrophages in mice with high efficiency and specificity. Using these novel nanoparticles, we achieved in situ functional identification of single macrophages in BM, liver, lung, and adipose tissues under conditions of acute or chronic inflammation. Moreover, with this intravital imaging platform, we further realized in vivo phenotyping of individual macrophages in the calvarial BM of mice under systemic inflammation. In conclusion, we established an efficient in vivo LD labeling and imaging system for single macrophage phenotyping, which will aid in the development of diagnostics and therapeutic monitoring. Moreover, this method also provides new avenues for the study of lipid trafficking and dynamics in vivo.