A new label-free electrochemical DNA (E-DNA) biosensor using a custom synthesized ferrocenyl (Fc) double-stranded DNA intercalator as a redox marker is presented. Single-stranded DNA (ssDNA) was co-immobilized on gold electrodes with 6-mecarpto-hexanol to control the surface density of the ssDNA probe, and hybridized with complementary DNA. The binding of the Fc intercalator to dsDNA was measured by differential pulse voltammetry. This new biosensor was optimized to allow the detection of single base pair mismatched sequences, able to detect as low as 10 pM target ssDNA with a dynamic range from 10 pM to 100 nM. DNA extracted from wastewater was analyzed by quantitative polymerase chain reaction targeting human-specific mitochondrial DNA (mtDNA). The aim of this approach is to enable the analysis of population biomarkers in wastewater for the evaluation of public health using wastewater-based epidemiology (WBE). The E-DNA biosensor was employed to detect human-specific mtDNA from wastewater before and after PCR amplification. The results demonstrate the feasibility of detecting human DNA biomarkers in wastewater using the developed biosensor, which may allow the further development of DNA population biomarkers for public health using WBE.