Peptide nucleic acids (PNAs) have attracted tremendous interest in the fabrication of highly sensitive electrochemical nucleic acid biosensors due to their higher stability and increased sensitivity than common DNA probes. The neutral pseudopeptide backbone of PNAs not only makes the PNA/DNA duplexes more stable but also provides many opportunities to construct ultrasensitive nucleic acid sensors. This review presents the details of various protocols for the construction of PNA-based electrochemical nucleic acid sensors. The crucial factors, origin, and development of PNA, immobilization methods of PNA probes and signal generation mechanisms, are discussed. This review aims to provide a reference for ultrasensitive PNA electrochemical biosensor preparation.