Abstract Leptospirosis is a re‐emerging bacterial zoonotic disease that affects both humans and animals, with a significantly higher incidence in tropical and sub‐tropical regions. Disease control, epidemiology, and surveillance rely on a One Health approach, as accurate detection can be applied to humans, animals, and the environment. This study represents the first attempt to develop a method for detecting the pathogenic Leptospira santarosai serovar Shermani based on the latex agglutination reaction. The serological activity of the antibody is examined to achieve a high titer of antibody before adsorption onto polystyrene particles. Using a pH medium of 6.8–7.8, total antibody adsorption of up to 3 mg m −2 is achieved. Particle agglutination is observed after incubating the antibody‐adsorbed PS with leptospiral culture for 4 min, revealing a detection limit of 1.7 × 10 2 leptospires mL −1 . Interestingly, the detection limit increased by 1000 times when observing agglutination using spectrophotometer. The test exhibits high specificity with Shermani and shows negligible cross‐agglutination with non‐pathogenic Leptospira and water‐borne bacteria. Agglutination testing in collected water samples from natural sources demonstrates a good correlation with culture technique. This simple and rapid leptospires agglutination detection method can be applied as a screening test in environmental, human, and animal specimens.