The diagnosis of Lyme borreliosis (LB) relies primarily on clinical evaluation supported by appropriate serologic testing in selected cases. Serology is recommended only in suspected disseminated LB, characterized by compatible clinical signs and history of tick exposure. In early localized disease such as erythema migrans, laboratory testing is unnecessary due to low sensitivity and the reliability of clinical diagnosis. A two-tiered testing algorithm remains the standard: enzyme-linked immunosorbent assay (ELISA) followed by immunoblot confirmation when ELISA results are positive or equivocal. For patients with symptoms lasting less than six weeks and negative initial results, serology should be repeated after three weeks. Only IgG are considered to confirm LB diagnosis. Intrathecal antibody synthesis is critical for diagnosing Lyme neuroborreliosis (LNB), achieving > 99 % sensitivity after 6–8 weeks, although isolated antibody index elevation without pleocytosis suggests alternative etiologies. Interpretation of serology must always consider clinical context: IgG may remain for years after recovery, and isolated IgM beyond six weeks typically represents a false positive. Serologic limitations include low sensitivity in early disease and cross-reactivity, particularly for IgM. PCR may aid diagnosis from synovial fluid or skin lesions but is rarely informative for cerebrospinal fluid. Emerging biomarkers such as CXCL13 and advanced molecular approaches remain experimental and require further validation.