Diagnosis of rickettsial infections is challenging due to nonspecific clinical symptoms and limitations of current diagnostic methods. Molecular assays allow early detection but are limited by cost and technical demands, whereas conventional serological tests often exhibit cross-reactivity and low sensitivity during the early stages of infection. This study aimed to develop and evaluate a recombinant-antigen sandwich ELISA for improved antibody detection against Rickettsia spp. Three Rickettsia akari proteins, rGroEL, rDnaK, and rA8GP63 (uncharacterized protein), were produced and validated for immunogenicity. The assay was evaluated using 94 patient serum samples, including those with positive, negative, and unknown clinical course. The optimized ELISA demonstrated high reproducibility, with IgG sensitivity of 89.47–95.39% and specificity of 90%. IgM detection, also assessed, showed lower sensitivity (42.11–82.89%) but maintained strong specificity (83.33%). The diagnostic performance was comparable to that of a commercial indirect immunofluorescence assay, with no cross-reactivity detected in sera from patients with unrelated infections. rDnaK and rA8GP63 represent newly explored diagnostic candidates. These findings highlight the potential of this recombinant protein-based ELISA as an accessible, sensitive and specific diagnostic tool, with a meaningful clinical impact for improving the early and accurate detection of rickettsial infections.