Aims: to develop a monoclonal antibody (MAb) for the rapid detection of Aeromonas hydrophila in human faeces. Methods and Results: A monoclonal antibody with strong specificity against Aer. hydrophila was obtained by the fusion of myeloma cells and splenocytes of a mouse immunized with vegetative cells of Aer. hydrophila ATCC 7966, followed by a two-step selection against other species of the genera. ELISA analyses revealed that MAb 5F3 strongly reacts with all the Aer. hydrophila strains evaluated, showing a just basal reactivity against other species of the genera, especially Aer. sobria and Aer. caviae . Conclusions: MAb 5F3 was characterized as an IgG that recognized a polypeptide of approximately 110 kDa. Significance and Impact of the Study: This MAb could be used to detect Aer. hydrophila in human stool samples.