Background: Babesia is an intraerythrocytic parasite that has always been misdiagnosed to malaria parasite, rendering to inappropriate treatment of the disease especially in co-endemic areas. Unlike Plasmodium species, intraerythrocytic cycles of Babesia including their invasion, growth, division, and egress are still not fully understood. In recent years, optical diffraction tomography (ODT) has shown great potential in the field of pathogen detection by virtue quantification of three-dimensional imaging tomograms. Methods: The early ring stage of P. falciparum and B. microti were obtained from the infected patients during diagnosis. The ODT technique was applied to analyze and characterize detailed differences at the single cell level between P. falciparum and B. microti ring stage. Based on three-dimensional quantitative information, accurate measurement was performed in morphological, biochemical, and biophysical parameters. Results: Accurate measurements indicated actual differences in morphological parameters in which the host cell surface area at the ring stage in B. microti was significantly smaller than P. falciparum, and sphericities showed higher levels in B. microti parasitized cells. Based on biochemical parameters, host cell hemoglobin level significantly increased and membrane fluctuation were more active fluctuating in P. falciparum than B. microti infected cells. The result indicates that P. falciparum more actively altered host RBCs. Conclusion: Although P. falciparum and B. microti showed often confusable characteristics under the microscopy, the actual three-dimensional properties are different. These differences could be used in differential clinical diagnosis of erythrocytes infected with B. microti and P. falciparum.