Paper-based devices hold great promise in biosensing, but the choice of electrode materials influences performance. Here, we report a paper-based electrochemical sensor developed for nucleic acid quantification, in a sandwich-type architecture integrating 3-electrode systems on metallized electrospun polymeric fibers. A 3D-printed hydrophobic barrier on the chromatographic paper defines injection and testing zones. Fluid diffusion through paper and concentration gradients are considered in the design. Electrochemical characterization is performed using 40 μL of methylene blue solution, which interacts with double-stranded nucleic acids, reducing its redox activity. This interaction mechanism within the paper substrate is confirmed by spectroscopy. The sensor achieves detection of nucleic acids in 3 min with 2 μL of solution. Real sample analysis is performed for the quantification of PCR-amplified genes with a limit of detection of 1.38 ng μL−1. The device serves as a promising point-of-care diagnostics tool for the direct quantification of amplified genetic material.