BACKGROUND Rhizome rot, caused primarily by Fusarium oxysporum, is one of the most destructive diseases leading to significant loss in ginger worldwide. The loss can be greatly reduced by proper disease management practices steered by accurate and early diagnosis of pathogens. Pathogen detection at an early stage of infection can also reduce the incidence of disease epidemics. Classical methods are often time consuming, relying on culturing the putative pathogens and the availability of expert taxonomic skills for accurate identification, which leads to the delayed application of control measures. The development of a simple, rapid, sensitive and cost-effective point-of-care diagnostic tool is thus one of the major research priorities for rhizome rot. RESULTS The 65 kDa, immunoreactive protein band was selected as a diagnostic marker and was subjected to MS analysis followed by blastp. Based on blast result, a synthetic antigenic peptide was synthesized, and used to generate pAbs. The peptide-specific antibodies were used to develop a colloidal gold immunochromatographic assay (ICA). The sensitivity, specificity, and accuracy of ICA were 92.59%, 81.25%, and 90%, respectively. The ICA has a visual detection limit of 2.122 μg mL-1 for infected rhizome samples and 5.065 μg mL-1 for leaf samples with optimal detection time within 5 min. Moreover, the ICA also detected early stage infected samples, of which 71.42% (50/70) were true positives. CONCLUSIONS Findings from this study indicated that the assay can be utilized as a tool for the investigation of rhizome rot infection in field samples. © 2019 Society of Chemical Industry.