Gold nanoparticles (AuNPs) can provide a simple, easy-to-use, inexpensive, at hand point-of-care (POC) fast to diagnose; however, AuNPs have the predisposition to form aggregations. Since the nanoparticles stability is an important issue, this article is aiming to study the long-term stability associated with the development of an immunosensor for clinical diagnosis. Here, we assessed two previous methods commonly described in the literature to prevent the formation of aggregate by studying pH and Tween® 20 (polysorbates) addition as surfactant. AuNPs were characterized through ultraviolet-visible (UV-Vis), dynamic light scattering (DLS) and transmission electron microscopy (TEM) and through analysis in the ImageJ software. We found that the Tween® 20 provided more than stable condition in aqueous solution in comparison to pH dependence. The fabricated AuNPs were further used to detect dengue virus and demonstrating that its use at pH 7.2 did not maintain reproducibility in the detection of dengue virus after one year. Unlike, the Tween® 20 modified AuNPs that detected dengue virus soon after the synthesis and over the course of one year demonstrating the high sensitivity of immunosensor. Finally, our result showed excellent dispersity throughout the year when using Tween® 20 to avoid aggregation.