Objective To perform differential diagnosis of a case of Babesia microti infection earlier misdiagnosed as malaria, and analyze the clinical treatment.Methods Clinical and epidemiological information of the case was collected. Blood sample was collected and examined by microscopy with. Giemsa-stained thick and thin blood smears for observing Babesia morphologically. Peripheral blood sample was also underwent rapid immunological assay for malaria parasite. DNA was extracted from the patient’s blood sample and PCR amplification was performed with genus and species-specific primers of Babesia 18S rRNA gene, and specific primers of Plasmodium falciparum small subunit ribosomal ribonucleic acid (SSU rRNA) gene.Results The patient had recurrent fever for more than 2 months since February 2019, and his blood routine examinations showed progressive decrease of white blood cells, red blood cells, and platelets, with moderate anemia and splenomegaly. Epidemiological investigation showed that the patient had no history of travel out of town. A large number of P. falciparum-like parasites were seen in the thick and thin blood smears of the patient, and and malaria rapid diagnostic assay presented negative result. Using B. microti-specific primers, PCR amplification indicates genus and species-specific positive bands of 400 bp and 1 600 bp length, respectively.Conclusion Combining the clinical information, epidemiological history, etiology and PCR analysis data, the case was diagnosed as B. microti infection.