This study reports the development and validation of recombinant Listeria adhesion protein (rLAP) as a bio-recognition molecule for the rapid and specific detection of Listeria monocytogenes. The LAP gene was cloned into a pET vector, expressed in E. coli BL21 (DE3) under IPTG induction, and purified as a 104 kDa His-tagged protein via Ni-NTA affinity chromatography (yield: 2.88 mg/mL). Protein identity and purity were confirmed by SDS-PAGE and Western blotting. A LAP-based sandwich ELISA using anti-Internalin A antibodies demonstrated high specificity for L. monocytogenes serovars 4b and 4e, with a detection limit of 10⁴ CFU/100 µL, and no cross-reactivity with L. innocua or other non-target bacteria. Additionally, a lateral flow immunoassay (LFA) was developed using rLAP as the test line antigen and gold nanoparticles (GNPs) conjugated with specific antibodies. GNPs synthesized via citrate reduction were characterized by DLS (31.11 nm, −37.5 mV) and UV-Vis spectroscopy. Optimized antibody-GNP conjugates (33.46 nm, −16 mV) and LAP-coated 8 µm nitrocellulose membranes enabled sensitive detection. The assay specifically detected L. monocytogenes strains (ATCC 19115, 19118, MTCC 1143) with no cross-reactivity to L. ivanovii, L. innocua, or other Gram-positive/negative bacteria. In spiked milk, after 24 h enrichment in LESM, the LFA achieved detection limits of 3.34 ± 0.01 log CFU/mL (broth) and 3.3 ± 0.02 log CFU/mL (milk), with results available in 20 ± 2 minutes. These findings establish LAP as a robust and biologically relevant capture molecule for developing rapid, cost-effective, and field-deployable immunodiagnostic platforms for food safety surveillance.