Abstract <p>Rotavirus is the main cause of acute diarrhea in children under 5 years old. Due to acute disease, diagnosis needs to be made at the point of care, even in low-resource settings. Furthermore, rotavirus has many circulating genotypes. Therefore, we aimed to develop a rapid test for rotavirus detection using polyclonal antibodies. The optimal conditions for generating the test strip were investigated. The detection antibody was conjugated to gold nanoparticles (20 nm in diameter) at pH of 9.0 and concentration of 100 mg/mL. The capture antibody was sprayed at the T-line of the nitrocellulose membrane at a amount of 12 μg/cm. The properties of the developed test strip were also evaluated. The test result was shown in 10–15 min. The detection limit of the test strip was determined to be as low as 5 × 10⁴ CFU/mL by a strip reader and 10⁶ CFU/mL by naked eyes. The test strip did not cross-react with 18 different potential interfering pathogens. Interestingly, the test strip can detect 5 predominant circulating rotavirus genotypes. The sensitivity and specificity of the test strip were evaluated by testing with 665 clinical samples and reached 95.26 and 99.51%, respectively. The Kappa statistics showed k-coefficient of 0.955, indicating a very good agreement between the developed test strip and ELISA results. From the findings, the developed test strip can be used for point-of-care rapid detection of rotavirus in fecal samples. This investigation can be considered the first work applying polyclonal antibodies for the development of a lateral flow immunoassay test strip to detect human rotavirus.</p>