Large-area, freestanding, two-dimensionally ordered macroporous (2DOM) Ag thin films with adjustable periodic spacings and good mechanical robustness were fabricated via nanosphere lithography at the solution surface (NSLSS) combined with interfacial reactions. The obtained 2DOM Ag thin films exhibited typical properties of plasmonic crystals with well-resolved reflectivity dips and vivid colors. The facile NSLSS method enabled the 2DOM Ag thin films to be readily transferred to arbitrary substrates to realize application as thin film sensors. Their application as both a surface plasmon resonance (SPR) sensor and a surface-enhanced Raman scattering (SERS) sensor in the detection of both small molecules and biological macromolecules were explored. As SERS substrates for the detection of 4-aminothiophenol (4-ATP) molecules, the 2DOM Ag thin films showed enhancement factors as high as the 107 order, which made them a promising sensor for the detection of trace amount of analyte adsorbed on the surface. As SPR sensors, the 2DOM Ag thin films modified with a self-assembled monolayer of recognizing molecules as binding sites showed remarkable shift in the reflectivity dips responsive to the chemical environments. When used for the specific detection of avidin molecules, the 2DOM Ag thin film biosensors exhibited excellent performance with low detection limit (≈100 pM) and broad working range (100 pM–200 nM), indicating that they may be a promising candidate for high-performance biosensors.