Diagnosis of tick-borne encephalitis (TBE) in patients is based on clinical findings, cerebrospinal fluid (CSF) analysis and findings of specific IgM and IgG antibodies in serum. In 15–20% of patients, diagnosis is possible only from second sample of serum 7–14 days after the onset of the second phase of the disease. Therefore, there is a need of fast, sensitive and accurate method for routine diagnostic of TBE in human samples. We have adapted our existing TBEV reverse transcription PCR (RT-PCR) detection system to the one-step qualitative real-time RT-PCR. The method is based on the detection of a nuclear acid amplification during exponential phase of PCR when the reaction reaches its optimal course. In our study we have worked with the LighCycler instrument (Roche Diagnostic, UK). As a detection format, DNA binding dye SYBR Green I have been used. Specificity and sensitivity of amplification reactions were enhanced by combining amplification with a melting curve analysis. The system was established and validated using viral RNA extracted from brain tissue of suckling mice (BALB/c) infected with TBEV (strain Hypr). In order to evaluate real-time RT-PCR for routine diagnostic purposes, we have applied the assay to CSF or serum samples from patients with early diagnosis of TBE (onset of fever, headache and neurological symptoms less than 3 days before admission to hospital and lumbal punction). Diagnosis of TBE was based on typical clinical picture and serological findings in serum sample (IgM over 150 WIEU, IgG over 150 WIEU). Of the 70 clinical samples investigated for TBEV RNA, none was tested positive. To avoid false negative results we have elaborated a positive control. We have spiked a negative patient CSF sample with 10-fold dilutions of viral RNA extracted from brain tissue of infected suckling mouse. Although the method was sensitive to detect 1 PFU in a reaction volume of microlitre of artificially infected CSFs, it fails to detect viral RNA in samples from patients with serologically proven TBE. The result indicates that virus load decreases with the onset of serum antibodies and cannot be regularly detected in CSF by RT-PCR. The method is not suitable for routine diagnosis of TBEV in patients with detectable antibodies and the suitability for detection in patients without serum antibodies should be evaluated.