Aims: The diagnosis of cat scratch disease (CSD), a disease caused by Bartonella henselae, is challenging and often hampered by the lack of appropriate laboratory assays in developing countries due to limited resources. Currently, the indirect immunofluorescence assay (IFA) is the mainstay for CSD diagnosis. However, IFA kits are costly as limited samples can be tested on one slide and reading of the immunofluorescence results is subjective. In this study, the sensitivity and specificity of a recombinant B. henselae outer membrane protein (BHp26)-based enzyme-linked immunosorbent assay (ELISA) was assessed for serodiagnosis purposes. Methodology and results: Bartonella henselae outer membrane protein (BHp26) gene was cloned into a pBAD-TOPO expression plasmid and transformed into a TOP10 Escherichia coli host. The recombinant protein BHp26 was purified using an affinity chromatography approach in an AKTA purifier 10 system. The immunogenicity of the purified recombinant protein was evaluated using Western blot (WB). A recombinant outer membrane protein-based enzyme-linked immunosorbent assay (ELISA) was developed for detection against B. henselae antibodies in human sera. The recombinant protein-based ELISA demonstrated 57.7% agreement and 25% sensitivity as compared to IFA. A high specificity (94%) was exhibited when the ELISA was tested against 50 patients’ sera with positive findings to other infectious causes, including dengue, rickettsiae, leptospira, legionella and mycoplasma. Using the ELISA developed in this study, 14% (7/50) of urban blood donors and 9.1% (5/55) of healthy individuals from rural areas had IgG antibodies detected against B. henselae, suggesting previous exposure to the pathogen. Conclusion, significance and impact of study In view of the rising incidence of CSD, the recombinant outer membrane protein-based ELISA will be helpful for screening a large sample size of human sera for serosurveillance study.