DNA detection based on peptide nucleic acid (PNA)-DNA hybridization is emerging as an important method in the area of DNA microarrays and biosensors because PNA shows remarkable hybridization properties. In this work, we provide a novel, simple, sensitive, and selective strategy based on a PNA-graphene oxide (GO) assembled biosensor for fluorescence turn-on detection of DNA, in which the new nanomaterial GO was used as a scaffold for PNA and a quencher for the fluorophore. The PNA-GO assembled biosensor is capable of distinguishing sequence specificity including complementary, one-base mismatched and non-complementary targets. Moreover, the results show that the biosensor is able to detect target DNA down to hundreds of picomolar. This sensing platform has been demonstrated to be highly sensitive and specific, and we expect that it will find great applications in the field of biomedicine and disease diagnostics.