We report for the first time the whole development of a biosensing system based on the Interferometric Optical Detection Method (IODM) enriched with gold nanoparticles (AuNPs), acting as interferometric enhancers for improving the performance of immunoassays. For this purpose, the Lactoferrin sandwich immunoassay model was employed. We describe in detail the entire value chain from the AuNPs production, its functionalization, and characterization with anti-Lactoferrin (anti-LF), the biosensing response of these conjugates as well as their corresponding calculation of the kinetic constants, performance comparison of the readout interferometric signals versus Scanning Electron Microscopy (SEM) and the percentage of the sensing surface covered. Finally, a Lactoferrin sandwich immunoassay was carried out and correlated with Enzyme-Linked ImmunoSorbent Assay (ELISA), and the Limit of Detection and sensitivity figures were obtained. As a result, we demonstrate how the AuNPs act as interferometric amplifiers of the IODM for improving the biosensing response, opening the possibility of being applied in multiple biological detection applications.