Surface Enhanced Raman Spectroscopy (SERS) method is widely used as a powerful undamaged tool to analysis low concentration of molecules, which gives great potential for bio-detection. In this work, large area SERS substrates were fabricated by hybrid large area porous GaN nanostructures with Au/Ag. By controlling porous GaN morphology, the Au/Ag layer thickness and morphology, the detection limit of 1.13 × 10-13 M and enhancement factor of 2.5 × 108 of t he large area SERS substrate were obtained with good uniformity and stability by using R6G as SERS probe. The high performance of the SERS substrate is attribute to the electromagnetic enhancement after hybrid GaN nanostructure with Au /Ag, which conformed by finite difference time domain method (FDTD) simulation. The potential application for the detection of miRNA (miR-K12-5-5p as breast cancer related biomarker) was evaluated with fluorescent molecular (FAM) as Raman target molecular and a sensitive miR-K12-5-5p detection with a limit down to 8.84 × 10-10 M was achieved, which confirming the potentialities of the Au/Ag/ porous GaN hybrid SERS substrate for early-cancer diagnosis, and giving great potential to develop ultrasensitive SERS biosensors for various biological and chemical analyses.