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Background: Tick-borne encephalitis (TBE) constitutes a public health concern in Europe. Certain coastal municipalities in southern Norway are considered TBE risk areas and in the last two years, there have been increasing numbers of TBE cases. Since the majority of infections are claimed to be asymptomatic, the aim of the current study was to assess the seroprevalence of antibodies to tick-borne encephalitis virus (TBEV) among unvaccinated adults living in a TBE endemic area in Norway., Methods: One thousand one hundred and twenty-three blood donors living in Vestfold and Telemark county were included and associated sera were analysed for TBEV IgG antibodies. Information regarding tick bites, previous flavivirus exposure and knowledge regarding TBE and TBE prevention were obtained through a questionnaire., Results: Fifty-eight samples were reactive by ELISA, of which 21 (36.2%) were confirmed by a TBEV-specific serum neutralization test. Of the 21 blood donors with neutralizing TBEV antibodies detected, 17 reported previous TBE vaccination. Thus, only four blood donors (0.4%) had TBEV neutralizing antibodies consistent with previously undergone TBEV infection. Regarding TBE awareness, half of the blood donors were familiar with TBE, but only 35% were aware of a preventive TBE vaccine., Conclusions: Our study indicates low prevalence of subclinical TBEV infections among blood donors living in Vestfold and Telemark county and there is a lack of awareness among general public.