In this paper, we present a class of bio-dots, polyepinephrine (PEP)-based fluorescent organic dots (PEP-FODs) for selective and sensitive detection of Fe2+, Fe3+, and Cu2+. The PEP-FODs were derived from epinephrine via self-polymerization at relatively low temperature down to 60°C with low cytotoxicity and relative long lifetime (7.24ns). The surface morphology and optical properties of the synthesized PEP-FODs were characterized. We found that the diameters of PEP-FODs were mainly distributed in the narrow range of 2–4nm with an average diameter of 2.9nm. An optimal emission peak located at 490nm was observed when the green light-emitting PEP-FODs were excited at 400nm. It is discovered that Fe2+, Fe3+, and Cu2+can strongly quench the fluorescence of PEP-FODs through the nonradiative electron-transfer. The detection limit of 0.16, 0.67, and 0.15μM was obtained for Fe2+, Fe3+, and Cu2+, respectively. The independent sensing platform of Fe2+, Fe3+, and Cu2+could be established by using NaF as a complexing agent and by regulating the reaction time between NaF and metal ions. Cell viability studies reveal that the as-prepared PEP-FODs possess good solubility and biocompatibility, making it as excellent imaging nanoprobes for intracellular Fe2+, Fe3+, and Cu2+sensing. The developed PEP-FODs might hold great promise to broaden applications in nanotechnology and bioanalysis.