Aquatic viruses cause devastating diseases in aquaculture, severely limiting production and resulting in significant economic losses. The giant salamander iridovirus (GSIV), a member of the genus Ranavirus, is the only virus reported to infect giant salamanders, posing a severe threat to the farming industry. Currently, there are no effective strategies available for its control. In this context, a reliable diagnostic tool for the rapid detection of GSIV is crucial to mitigate its impact. This study developed a nanobody-based immunoassay for the rapid and reliable detection of GSIV. GSIV was cultured and used to immunize an alpaca, A phage library with an original diversity of 1.89 × 108 PFU was established, and six nanobodies were identified after three rounds of panning. Among them, HC-2 exhibited superior performance and was used to develop a highly sensitive ELISA method employing streptavidin-PolyHRP (SA-PolyHRP) as a signal amplification strategy. The assay achieved a detection limit of 3.3 × 105 PFU/mL and demonstrated high specificity without cross-reactivity. Practical application was validated in infected giant salamander samples, underscoring its diagnostic potential. This work provides a robust tool for GSIV diagnosis and showcases the potential of nanobodies in advancing aquaculture diagnostics.