Ultrastable aqueous graphite nanofluids were prepared by single-step liquid-phase pulsed laser ablation (LP-PLA), and they showed an outstanding long-term stability without any agglomeration, even though no surfactant was used. It was revealed with FT-IR spectroscopy that the hydrophilic functional groups were introduced onto the surface of graphite nano-particles and that such hydrophilic nature showing a high negative potential was the origin of the outstanding long-term stability. Finally, aqueous graphite nanofluids demonstrated an enhanced thermal conductivity by 1.49% with low concentration (0.02 vol %), compared to pure water.