Infectious diseases are a serious problem and a major contributor to severe economic losses in intensive fish culture. Therefore, rapid and sensitive detection of fish pathogens is extremely important. Although various assays for determination of fish pathogens have been developed, most of these diagnostic methods are time-consuming and laborious. To overcome these limitations, functional nanomaterials have been actively investigated to improve detection ability and rapidity of diagnostic assays. Gold nanoparticles (AuNPs) have been widely studied for their unique optical properties arising from their surface plasmon resonance, which is responsible for their large absorption and scattering properties. These unique properties are four to five orders of magnitude larger than those of conventional dyes and can be controlled by varying their sizes, shapes, and compositions. Moreover, AuNPs can be easily synthesized and functionalized with different biomolecules, including pathogen-specific oligonucleotides or antibodies. Recently, nanoparticle-based assays have been introduced as a tool for laboratory diagnosis. They have been used for the direct detection of unamplified nucleic acids in hybridization assays. Single- and double-stranded oligonucleotides can be adsorbed on AuNPs in colloidal solution under certain conditions. The result of the hybridization process can be visually detected within 1 min after addition of AuNPs, when the color of the reaction mixture changes from red to blue (positive reaction) or remains red (negative). The development of such nanoparticle-based strategies holds the potential to become powerful approaches for diagnosis of fish pathogens.