Lysozyme (LYZ) plays a crucial role in the body's immune defense system. Monitoring LYZ levels can provide valuable insights into the diagnosis and severity assessment of various diseases. Traditionally, antibody-based sandwich assays are employed for LYZ detection, but they are often time-consuming and operationally complicated. In this research, a novel sandwich FRET biosensor was developed, which enables rapid detection of LYZ based on peptide-functionalized gold nanoparticles (pAuNPs) and FAM-labeled aptamer (Apt-FAM). Initially, a mixture of Apt-FAM and pAuNPs resulted in partial quenching of the Apt-FAM fluorescence emission through an inner filter effect (IFE), with negligible energy transfer because of the electrostatic repulsion between the negatively charged pAuNPs and Apt-FAM. The introduction of LYZ into the mixture drove the specific binding of Apt-FAM and pAuNPs to LYZ, facilitating the formation of a pAuNPs-LYZ-aptamer sandwich structure. The formation of this complex drew the pAuNPs and Apt-FAM into close enough proximity to enable FRET to occur, which in turn effectively quenched the fluorescence emission of FAM. The decrease in FAM fluorescence intensity was correlated with the increasing concentration of LYZ. Thus, a sandwich FRET biosensor was successfully developed for LYZ detection with a linear detection range of 0–1.75 μM and a detection limit of 85 nM. Additionally, the biosensor allowed visual detection of LYZ in a 96-well microplate, with a rapid response time of just 15 s. This study introduces a innovative sandwich FRET biosensor that combines aptamer and peptide recognition elements, offering a fast and antibody-free method for protein detection.