In this study, we synthesized novel gold carbon dots (GCDs) with unique properties by microwave assisted method. The characterization of high-resolution transmission electron microscope (HRTEM), XRD, high angle annular dark field scanning transmission electron microscope (HAADF-STEM), and energy dispersive spectrometer demonstrates that GCDs are composed of carbon and Au. Tiny Au clusters are dispersed in a 2 nm-size carbon skeleton, which integrates the properties of typical CDs and gold nanoclusters (AuNCs), displaying fascinating peroxidase-like activity and single excitation/dual emission. Dual emission of the GCDs exhibits different fluorescent response to the target species and enables the GCDs to be exploited for sensing and bioimaging. The highly photostable and biocompatible GCDs were applied to dual fluorescent imaging for breast cancer cells and normal rat osteoblast cells under a single excitation. Moreover, ratiometric fluorescence imaging was used to monitor Fe3+ level in normal rat osteoblast cells.