Nitrogen rich carbon nanoparticles are known to provide higher fluorescence stokes shift, and thereby are potential candidates for fluorescent sensors. Herein, a facile one-step hydrothermal synthesis is reported for N-rich carbon nanospheres (G-CNS) from caffeine and o-phenylenediamine as precursors. The as-synthesized G-CNS showed high fluorescence with λem at 509 nm, with a highly selective fluorescence turn-off response towards Fe2+/Fe3+, rendering these carbon nanospheres as potential candidates to detect intracellular labile iron pool in live cells. The intracellular labile iron pool in iron-overloaded cells was sensed using the synthesized G-CNS. Mechanistically, the fluorescence quenching via dynamic pathway involves the formation of an excited state charge transfer process, which undergoes non-radiative decay.