This study presents the development and characterization of Au-Se nanoprobes (NPs) for the specific and simultaneous detection of matrix metalloproteinase-1 and -12 (MMP-1/12), key biomarkers in non-small-cell lung cancer (NSCLC). Leveraging the stability of Au-Se bonds against biological thiols, such as glutathione (GSH), the NPs exhibit remarkable resistance to interference, maintaining its fluorescence signal across a wide range of temperatures, pH levels, and in the presence of high concentrations of GSH. The synthesized NPs demonstrate high specificity and sensitivity toward MMP-1/12