Based on an immobilization-free electrochemical DNA detection platform, a novel electrochemical method for detecting and monitoring the activity of DNA polymerase has been developed. The determination of the existence of DNA polymerase is accomplished through the detection of a DNA primer which is elongated along a DNA template under the catalysis of the DNA polymerase. The detection utilizes the competition between the elongated DNA and a ferrocene-labeled peptide nucleic acid probe (Fc-PNA) to hybridize with the DNA template in solution. The neutral PNA backbone and the electrostatic repulsion between the negatively charged DNA backbone and the negatively charged electrode surface are then exploited to determine the result of the competition through measurement of the electrochemical signal of Fc. In the presence of the DNA polymerase, the DNA primer is extended to the same length as the DNA template, resulting in stronger hybridization affinity between the two complementary DNA strands, which releases the Fc-PNA from the Fc-PNA/template hybrid and allows it to freely diffuse to the negatively charged electrode to produce a significantly enhanced electrochemical signal of Fc. This simple and robust electrochemical DNA polymerase assay does not require any probe immobilization steps and could potentially benefit drug development research, as well as on-site pathogen monitoring applications.