The biological effect of endogenous food-borne carbon dots (CDs) is one of the controversial issues of current research areas in food biotechnology. In this study, the biocompatibility and biofunctionality of sugar beet molasses-derived fluorescent carbon dots (CDs) were investigated for the first time in mammalian macrophage and fibroblast cells. The molasses CDs were nearly spherical in shape and monodispersed with a typical amorphous structure and a particle size distribution in the range of 1.3–3.8 nm. The anionic molasses CDs could easily enter the cells and exhibited excellent biocompatibility. Importantly, CDs with high photostability not only enabled the intracellular tracking of the nanomaterials by confocal microscopy, but also could reduce the LPS-induced NO production in RAW 264.7 cells. Altogether, endogenous CDs with dual functionality, bioimaging and anti-inflammatory effect, are believed to have great potential as macrophage-mediated theranostic agents.