A reduced graphene oxide (R-GO)-based field-effect transistor (FET) biosensor used for ultrasensitive label-free detection of DNA via peptide nucleic acid (PNA)-DNA hybridization is reported. In this work, R-GO was prepared by reduction of GO with hydrazine, and the FET biosensor was fabricated by drop-casting the R-GO suspension onto the sensor surface. PNA instead of DNA as the capture probe was employed, and DNA detection was performed through PNA-DNA hybridization by the R-GO FET biosensor. The detection limit as low as 100 fM was achieved, which is 1 order of magnitude lower than that of the previously reported graphene FET DNA biosensor based on DNA-DNA hybridization. Moreover, the R-GO FET biosensor was able to distinguish the complementary DNA from one-base mismatched DNA and noncomplementary DNA. Interestingly, the fabricated DNA biosensor was found to have a regeneration capability. The developed R-GO FET DNA biosensor shows ultrasensitivity and high specificity, indicating its potential applications in disease diagnostics as a point-of-care tool.