Affinity sensing of nucleic acids is among the most investigated areas in biosensing due to the growing importance of DNA diagnostics in healthcare research and clinical applications. Here, we report a simple electrochemical DNA detection layer, based on poly-l-lysine (PLL), in combination with gold nanoparticles (AuNPs) as a signal amplifier. The layer shows excellent reduction of non-specific binding and thereby high contrast between amplified and non-amplified signals with functionalized AuNPs; the relative change in current was 10-fold compared to the non-amplified signal. The present work may provide a general method for the detection of tumor markers based on electrochemical DNA sensing.