Detecting and identifying single molecules are the ultimate goal of analytic sensitivity. Single molecule detection by surface-enhanced Raman scattering (SM-SERS) depends predominantly on SERS-active metal substrates that are usually colloidal silver fractal clusters. However, the high chemical reactivity of silver and the low reproducibility of its complicated synthesis with fractal clusters have been serious obstacles to practical applications of SERS, particularly for probing single biomolecules in extensive physiological environments. Here we report a large-scale, free standing and chemically stable SERS substrate for both resonant and nonresonant single molecule detection. Our robust substrate is made from wrinkled nanoporous Au₇₉Ag₂₁ films that contain a high number of electromagnetic "hot spots" with a local SERS enhancement larger than 10⁹. This biocompatible gold-based SERS substrate with superior reproducibility, excellent chemical stability and facile synthesis promises to be an ideal candidate for a wide range of applications in life science and environment protection.