Since an indirect fluorescence immunoassay (IFA) for the detection of specific antibodies against Babesia divergens in human sera is not commercially available, an in-house prepared B. divergens IFA for the examination of bovine sera was established for serological studies in humans. To determine whether the described IFA is appropriate for such studies, 2 B. divergens antigens (of human or bovine origin) were tested using serum samples obtained from febrile human patients with a history of ‘tick bite’. Sera from other species of animals infected with B. divergens, Babesia EU1, B. microti or B. canis were also included for comparative purposes. All serum samples were also tested using a commercially available IFA for the detection of antibodies against B. microti, and the results compared with those obtained using blood smear and molecular techniques. This study showed that the evaluation and standardization of a B. divergens IFA for testing human sera is critical and that different B. divergens antigens provide different end-point titres of antibodies, leading to false negative or positive results. Serological cross-reactivity between B. divergens and Babesia EU1 needs to be taken into account when interpreting IFA results.