This study presents a novel method for DNA detection with multi-layer AuNPs to enhance overall detection sensitivity. This essay achieves not only an innovative radio-frequency biosensor but also a critical signal amplification methodology. Results show that bandwidth change for multi-layer AuNP with hybridization of DNA exceeds that for the double-layer AuNP up to 0.5 GHz. Furthermore, the developed biosensor detection limit for the DNA set employed in this essay is currently 10 pM. A single base-pair mutation of the wild-type target DNA could be distinguished from the perfect match target DNA at the melting temperature of 47 degrees C with a temperature controlling system. Experimental results in this study indicate that the proposed biosensor and the developed amplification methodology are successful. As health care becomes much more essential in modern life, this biosensor has potential applications in a screening kit for recognizing, sensing, and quantifying biomolecules in real samples.