The early diagnosis of major diseases, such as malignant tumors, has always been an important field of research. Through screening, early detection of such diseases, and timely and effective treatment can significantly improve the survival rate of patients and reduce medical costs. Therefore, the development of a simple detection method with high sensitivity and strong specificity, and that is low cost is of great significance for the diagnosis and prognosis of the disease. Electrochemical DNA biosensing analysis is a technology based on Watson Crick base complementary pairing, which uses the capture probe of a known sequence to specifically recognize the target DNA and detect its concentration. Because of its advantages of low cost, simple operation, portability, and easy miniaturization, it has been widely researched and has become a cutting-edge topic in the field of biochemical analysis and precision medicine. However, the existing methods for electrochemical DNA biosensing analysis have some shortcomings, such as poor stability and specificity of capture probes, insufficient detection sensitivity, and long detection cycles. In this review, we focus on improving the sensitivity and practicability of electrochemical DNA biosensing analysis methods and summarize a series of research work carried out by using electrically neutral peptide nucleic acid as an immobilized capture probe.