Advancing electrochemical sensors requires materials that offer both a high surface area and tailored surface properties. In this study, we developed mesoporous gold–silver–copper (AuAgCu) alloy films with a finely tuned composition and internal strain to boost their electrocatalytic activity. These multimetallic films were synthesized using a soft-template approach, in which self-assembled polymer micelles guided the formation of well-defined porous architectures on gold-sputtered glass substrates. By adjusting the relative amounts of gold, silver, and copper during fabrication, we achieved a balanced alloy structure that promotes defect formation and enhances electron transport. Among the compositions tested, the Au₀.₆Ag₀.₂Cu₀.₂ alloy exhibited the most favorable electrochemical performance. Characterization techniques including transmission electron microscopy and X-ray photoelectron spectroscopy confirmed a uniform alloy distribution and the presence of crystal imperfections—such as twin boundaries—that served as additional active sites. Based on follow-up analysis of chronoamperometric data from our ACS Nano-accepted study, the final limit of detection (LOD) for mesoporous AuAgCu films was determined to be 40.1 µM, significantly outperforming mesoporous Au (104 µM) and AuAg (447 µM) under identical, non-enzymatic sensing conditions. Notably, this enhancement was achieved without amplification strategies, photopatterning, or covalent functionalization. These features enabled effective detection of model analytes such as glucose, suggesting strong potential for biosensing and diagnostic applications. In conclusion, this work presents a practical strategy for designing mesoporous multimetallic films with enhanced surface properties and offers valuable insight into atomic-scale tuning of material compositions for improved performance in real-world sensing systems.