Localized surface plasmon resonance (LSPR)-based nano-biosensors are of great interest in various applications such as environmental protection, biotechnology, and food safety. It is well known that types of materials and shapes of the nanostructures have significant influence on the sensitivity and the resolution in nanobiosensor application field. The conventional nanostructure array used for nanobiosensor is fabricated by chemical method, so it is difficult to control the shape and the inter-particle space. Focused ion beam (FIB) can be used to produce any shapes with a very high level of regularity and reproducibility. This makes it possible to optimize the shape of nanostructures and the inter-particle distance to achieve the full potential application in nanobiosensor. Using focused ion beam (FIB) nanofabrication method, we have fabricated the regularly shaped hybrid Ag-Au nanostructures distributed on the surface of the glass substrate. Gold-coating was used to tuning the optical characteristics of the silver nanostructure array. Discrete dipole approximation (DDA) method was used to optimize geometrical parameters of the hybrid Ag-Au nanostructures. The design and experiment results show that the sensitivity and resolution have been improved considerably compared to the chemically synthesized nanostructures. The nano-biosensor demonstrates the potential applications in monitoring, detection and identiﬁcation of biological agents, and characterization of intermolecular interactions.