Sequence-specific detection of double stranded DNA (dsDNA) is important in various research fields. In general, denaturation of dsDNA into single strands is necessary for the sequence-specific recognition of probes to target DNA, posing several drawbacks which decrease the efficiency as a DNA sensor. Herein, we report a direct, sequence-specific dsDNA detection system without requiring any thermal denaturing step. Our strategy utilizes peptide nucleic acid (PNA) and graphene oxide (GO) as a probe and as a fluorescence quencher, respectively. The PNA first binds to the end of dsDNA strand due to the relatively easily dissociable terminal base pairs of DNA duplex. Next, superior binding affinity of PNA towards complementary DNA induces branch migration for gradual strand replacement, resulting in the formation of PNA/DNA duplex. Unlike other dsDNA sensors based on complementary DNA probes, PNA in combination with GO enabled hybridization with the target sequence hidden as a duplex form without denaturing step and thus, the formation of PNA/DNA duplex was translated into selective fluorescence signal. Moreover, it provided tighter turn-on signal control with very low background signal and high sensitivity and sequence selectivity even in the presence of serum proteins.