We set out to investigate the serological response of TBE virus (TBEV)-specific IgM and IgG antibodies in stored serum and cerebrospinal fluid (CSF) in notified TBE patients, in order to confirm or reject the diagnosis. We applied the ELISA methods used in clinical practice, Enzygnost and Immunozym, and assessed RT-PCR as a diagnostic tool. A total of 173 TBE cases were notified to the Public Health Agency. Samples from 129 patients were eligible for the study. Stored serum samples were found for 111 patients and CSF samples for 88 patients. All serum samples were analyzed with both Enzygnost and Immunozym, as well as an additional 140 control samples. CSF samples, including samples from ten controls, were analyzed with Immunozym. RT-PCR for TBEV was performed on 126 serum, two whole blood, 96 CSF, two feces and four nasopharynx samples. Only two of 111 notified patients lacked detectable TBEV IgM in serum, from whom one sample was RT-PCR positive. According to the ECDC definition, 117/129 (90.7%) of the reported TBE cases were confirmed. Positive RT-PCR results were obtained in eight patients, one from whole blood and eight from serum samples. Four out of eight of the RT-PCR positive patients were TBEV-IgM positive and none had detectable TBEV-specific IgG. All of the tested CSF, feces and nasopharynx samples were RT-PCR-negative. TBEV-specific IgG was detected in 88.4% and IgM in 31.6% of the CSF samples. RT-PCR on serum samples and CSF IgG antibodies can be used as complementary methods in TBE diagnostics, not least early in the disease course.