Abstract: To detect Alternaria longipes in tobacco fields rapidly, the monoclonal antibody against A. longipes was labelled with colloidal gold, and the rapid test strips for detecting A. longipes based on lateral flow immunoassay were developed for the first time. The results suggested that the test strips had a detection limit of 20 ng/mL and had no cross-reactivity with other tobacco-associated phytopathogens, which indicated its high sensitivity and specificity. Spiked fresh tobacco leaves were examined by using the test strips and the result could be obtained within 10 min. The colloidal gold strips are easy in operation and can be used for the rapid detection and early warning of occurrence of brown spot disease in tobacco fields.