Immunoassays are biochemical analytical techniques based on the specific binding between antigens and antibodies. Due to their high sensitivity, rapid analysis, high throughput, cost-effectiveness, and broad applicability, immunoassays have been widely utilized across various fields. Antibody–enzyme conjugates are essential components for signal generation in immunoassays, with crosslinking chemical (crosslinker)-based methods traditionally employed for their production. However, these methods often involve hazardous reagents and complex procedures, highlighting the need for more eco-friendly alternatives. In this study, we developed a universal, crosslinker-free approach for preparing antibody–enzyme conjugates. This method eliminates the use of chemical reagents such as glutaraldehyde or periodate, as well as the intricate conjugation and purification steps typically required. Instead, gold nanoparticles (AuNPs) were employed as a versatile nanomaterial to passively adsorb horseradish peroxidase (HRP) and antibodies through a simple procedure completed within 60 minutes. The resulting conjugates were characterized using UV-vis spectroscopy, transmission electron microscopy (TEM), and zeta potential analysis. Using acetamiprid (ACE), a widely-used pesticide, as the target model, an enzyme-linked immunosorbent assay (ELISA) was developed based on the AuNP-mediated conjugates. The assay demonstrated performance comparable to that of traditional crosslinker-based methods, with an IC50 of 0.49 ng mL−1 and a limit of detection (LOD) of 0.11 ng mL−1. Recovery rates for ACE in spiked fruit samples were consistent with those obtained using liquid chromatography-mass spectrometry (LC-MS). The proposed method not only provides a reliable alternative for HRP and antibody conjugation but also advances green analytical chemistry by minimizing the use of hazardous chemicals, reducing derivative waste, and improving energy efficiency.