We have investigated peptide nucleic acid (PNA)-mediated aggregation of reduced graphene oxide (rGO) sheets. Addition of PNA into suspension of rGOs resulted in aggregation of rGOs, which could be easily detected with the naked eye. To elucidate the mechanism of rGO aggregation, we monitored the reaction by transmission electron microscopy, zeta potential measurement, and UV-vis spectroscopy. Our findings suggest that PNA adsorbed on the rGO surface and then acted as a cross-linker to induce aggregation. We also tested the effects of different nucleic acids on rGO aggregation and found that not only the single-stranded DNA, but also the PNA-DNA complex, could stabilize the suspension against aggregation through electrostatic repulsion. Based on our understanding on rGO aggregation, we attempted to detect mutations in epidermal growth factor receptor (EGFR) by designing PNA probe to be complementary to the mutant type DNA sequence. Our results showed that PNA-mediated rGO aggregation could successfully be used to detect mutations in EGFR with high selectivity and sensitivity.