In this report, we summarized our development of biosensors for Rhodamine 6G and in vitro glucose detection based on surface-enhanced Raman scattering technology. For the detection of both Rhodamine 6G and in vitro glucose, a nature-patterned substrate with gold films over nanostructures (NPS-AuFON) was used as the surface-enhanced Raman scattering sensor platform. The enhancement factor was calculated at 9 × 107. In the processing of the substrate, cyclic voltammetry was used to form nano-gold particles under different conditions. The Rhodamine 6G and glucose detection were then achieved on this substrate. Furthermore, we combined the potentiostatic technique and electrochemical adsorption to best detect glucose in low concentrations. The glucose oxidation potential (100 mV) was used to capture glucose close to the surface of the NPS-AuFON. The quantitative detection of glucose in solution and in situ inspection were confirmed. Further, we determined that this surface modification technology can reach the goal of experiments set by the World Health Organization to judge whether or not a patient is a diabetic by detecting a glucose concentration of 11.1 mmol/L (mg/dL) at a minimum.