The diagnosis of Lyme borreliosis is based on a combination of clinical, epidemiological, and biological evidence. From a biological standpoint, serology is the primary diagnostic tool. A sequential combination of two techniques—ELISA followed by Western blot—provides the best performance in terms of sensitivity and specificity for the detection of specific antibodies. A thorough understanding of the principles and limitations of these techniques, as well as the kinetics of the immune response at different stages of infection with Borrelia burgdorferi sensu lato, enables a correct interpretation of test results. Thus, in the early cutaneous stage of infection (erythema migrans), a negative serology does not rule out the diagnosis. Conversely, especially in cases of nonspecific symptoms, a positive serology does not necessarily indicate an active infection, nor, due to the long-term persistence of antibodies, the definitive cause of these symptoms. Other tests with poor performance or insufficient evaluation have been proposed. Their lack of specificity is not ethically acceptable in clinical laboratory medicine.