Tick-borne encephalitis (TBE) virus is the most important flaviviral etiological agent affecting the CNS in Europe and Asia, where it has a significant impact on public health. Current laboratory diagnosis is based mainly on the detection of specific IgM and IgG antibodies in serum and cerebrospinal fluid by serological methods. However, recent developments in PCR methods could reveal that molecular diagnostics may play a more important role than previously described, especially for the early differential diagnosis of TBE. This review considers the recent developments in TBE diagnostics. The advantages and disadvantages of both serological and molecular methods are presented. Moreover, the results of quality control assessment studies for serological and PCR diagnosis of TBE infections are discussed, showing the need for some laboratories to improve their test systems with regards to sensitivity and specificity. Both diagnostic techniques will continue to be valuable approaches in clinical diagnosis and TBE research.