Malaria is a serious but preventable and treatable infectious disease that is found in over 100 countries around the world. Correct and rapid diagnosis of malaria infection can rescue the patient of getting sicker and reduces the risk of disease spreading among humans. Chlamydomonas reinhardtii chloroplast is an attractive platform for expressing malaria antigens because it is capable of folding complex proteins, including those requiring disulfide bond formation, while lack the ability to glycosylate proteins; a valuable quality of any malaria protein expression system, since the Plasmodium parasite lacks N-linked glycosylation machinery. In this study, Cell-traversal protein for ookinetes and sporozoites (CelTOS) antigen from Plasmodium falciparum was expressed in the chloroplast of C. reinhardtii and a highly sensitive and specific indirect ELISA test was developed using C. reinhardtii expressed PfCelTOS to detect malaria. Results obtained demonstrated that expressed recombinant PfCelTOS accumulates as a soluble, properly folded and functional protein within C. reinhardtii chloroplast and indirect ELISA using sera from malaria-positive donors suggested the potential use of expressed PfCelTOS as a malaria antigen for diagnosis tests.