Recently, in vitro diagnostics (IVD) has been widely applied in the qualitative and quantitative analyses of medical diagnoses and drug development in biomedical technology. There are ~50% global IVD markets including immunoassay, molecular diagnostics, point-of-care testing, and biosensor detection. Therefore, the development of a manufacturing process technology for biosensor detection that is cost-effective and highly sensitive is important. In general, the biochip development of metal nanostructure arrays adopting localized surface plasma resonance (LSPR) is based on mechanical nanolithography with a tip of an atomic force microscope. However, it does not improve the irregular shapes and dimensions during the mass production of biochips, and does not increase the process area, so sensitivity is sacrificed. In this paper, we present a novel dual-wavelength laser direct-write nanolithography system for fabricating periodic gold nanoparticle arrays on substrates for biosensors. The substrates were immersed by a wet etching method after exposure. Isotropic etching using oxygen-plasma method transfer to the substrates was then performed to fabricate a suitable undercut in the lift-off of the dry etching process. The pitch, depth, and width of the periodic gold nanoparticle arrays were 500, 43, and 197 nm, respectively, and the measured transmission spectrum was 800 nm. Chips based on the periodic gold nanoparticle arrays were designed for LSPR biosensors with high sensitivity, to be widely applied in biomedical fields.