Although the analyses of HBV genomic DNA have traditionally been performed with commercial techniques, the high cost and long time consumed have hindered their applications in routinely diagnosis and prognosis of infection. We construct peptide nucleic acid (PNA) piezoelectric biosensor for real-time monitoring of hybridization of hepatitis B virus (HBV) genomic DNA. The PNA probe can combine to target DNA sequences more effectively and specifically than a DNA probe. The PNA probe was designed and immobilized on the surface of the biosensor to substitute the conventional DNA probe for direct detection of HBV genomic DNA without previous amplification by PCR. The hybridization assay was completed in 50 min. The detection limit was 8.6 pg/L and the clinical specificity was 94.44% compared with real time-PCR (RT-PCR). The PNA probe was able to distinguish sequences that differ only in one base. Detection sensitivity can be improved and detection time can be decreased by adding RecA protein-coated complementary ssDNA which complement to HBV gene regions. The QCM system we designed has the advantages of being rapid, label-free and highly sensitive and can be a useful supplement to commercial assay methods in clinical chemistry.