Aeromonas hydrophila is an emerging aquatic pathogen, widely distributed in the environment The ubiquitous aquatic pathogen A. hydrophila is responsible for motile aeromonas septicemia, causing mortalities among different fish species worldwide. A. hydrophila is naturally present in the fish gut; however, it infects the host usually during the period of stress. Thus, accurate and rapid identification of the causative agent is important for the treatment and control of this disease. Current diagnostic tools rely on microbiological isolation and identification of A. hydrophila, in addition to the use of PCR for the detection of some virulence factors of the isolated bacterium. Since these techniques are laborious, time-consuming and expensive, so in this study, we describe here the development of a rapid, specific and sensitive assay for detection of A. hydrophila DNA without prior amplification utilizing a specific probe conjugated with gold nanoparticles. A specific oligonucleotide probe based on the A. hydrophila aerolysin gene was designed for the Aeromonas-AuNPs-probe assay and the assay was optimized to detect only the DNA of A. hydrophila. The results of this study demonstrated that the developed Aeromonas-AuNPs-probe assay could enable the visual detection of A. hydrophila DNA within 30 min without any prior amplification, at a detection limit of 10.9 ng. Our study validated this assay as a rapid and specific tool for detection of A. hydrophila DNA directly from clinical fish samples without prior amplification.