Tick-borne encephalitis (TBE) is a zoonotic disease, transmitted mainly by the bite of ticks. The TBE virus (TBEV) belongs to the family Flaviviridae, genus Flavivirus and is able to cause meningoencephalitis. For serological TBEV detection, the neutralization test (NT) is the most specific assay available. Different NT protocols are used in the laboratories, and until now the performance of these NTs has never been tested in an external quality assessment (EQA). In this EQA, we compared the results of eight European laboratories in detecting 17 samples (11 TBEV positive, five flavivirus cross reactive, and one negative sample) by NT. Furthermore, 14 of these EQA samples and 15 additional samples were tested in different commercial assays: 15 immunoglobulin G (IgG) enzyme-linked immunosorbent assays (ELISAs) and an immunofluorescence assay (IFA). Four laboratories showed a good NT EQA performance, whereas four laboratories had some sensitivity problems. Additionally, two of these laboratories showed a lack in specificity, misidentifying a dengue-positive sample as TBEV positive. The comparison of the commercial ELISAs revealed a high sensitivity in all assays, but as expected for IgG, the ELISAs showed a high degree of flavivirus cross reactivity. The assessment of Vienna Units in some of the ELISAs revealed deviations in the standards used by the different companies. Therefore, these standards should be revised. Generally, in this EQA, we found that reliable NT protocols are used in most of the laboratories, and the evaluation of the IgG ELISAs and the IFA showed a good agreement.