In this work, we developed a simple and accurate peptide nucleic acid (PNA)-based sandwich hybridization assay for single nucleotide polymorphisms (SNPs) in the p53 gene. Our approach combines the enzyme-free toehold-mediated strand displacement reaction (SDR) with real-time enzyme-linked immunosorbent assay (ELISA) to detect SNPs with high sensitivity and specificity. A PNA-DNA heteroduplex with an external toehold is designed and fixed on well surface of a 96-well plate. The strand displacement from PNA-DNA heteroduplexes is initiated by the hybridization of target sequence with the toehold domain and ends with the fully displacing of the incumbent DNA. Finally, the as formed PNA-target DNA duplex with overhang at its 5′-end hybridizes with a biotin-labeled reporter PNA to form a sandwich structure on surface for signal amplification. The proposed PNA-based sandwich biosensor displays high sensitivity and greatly enhanced discriminability to target p53 gene segments against single-base mutant sequences compared to its all-DNA counterpart. Furthermore, the probe design is elegantly simple and the sensing procedure is easy to operate. We believe that this strategy may provide a simple and universal strategy for SNPs detection through easily altering the sequences of probes according to the sequences around target SNPs.