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Rapid and user‐friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on‐site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual‐sample microfluidic chip integrated with a real‐time fluorogenic loop‐mediated isothermal amplification assay (dual‐sample on‐chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on‐chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10–1 pg/μl for genomic DNA of tested bacteria and 10–4 to 10–5 pg/μl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time‐to‐positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on‐chip LAMP assay provides an effective dual‐sample and multiple pathogen analysis, and thus would be applicable to on‐site detection and routine monitoring of multiple pathogens in aquaculture. [ABSTRACT FROM AUTHOR]