Tick-borne encephalitis virus (TBEV) is responsible for the most serious human viral tick-borne disease in Europe. A greater knowledge of the natural foci of TBEV and their fluctuating characteristics is essential to better assess the temporal variations of TBEV cycle’s epidemiological risks. To describe the seasonal and inter-annual variations of the TBEV-cycle epidemiological parameters related to TBEV nymph-to-larva transmission, exposure of small mammals to TBEV and tick aggregation on small mammals, a longitudinal survey was conducted over a 3-year period to monitor a TBEV focus in a mountain forest in Alsace, eastern France. We trapped small mammals and collected questing ticks five times per year from 2012 to 2014. TBEV prevalence in questing nymphs was lower in 2013 than in 2012 and 2014, probably because small mammals (Myodes glaerolus and Apodemus flavicollis) were more abundant in 2012, reducing tick aggregation and co-feeding transmission between ticks. This prevalence was higher in autumn than spring, which suggests that nymph-to-larva transmission of TBEV mainly occurs in spring. Despite these variations in prevalence, the density of infected questing nymphs was constant over time, leading to a constant risk for humans. The seroprevalence of small mammals was also constant over time although the proportion of small mammals harbouring ticks varied between years and seasons. Our results draw attention to the importance of considering the complex relationship between small mammal densities, tick aggregation on small mammals, density of infected questing nymphs and prevalence of infected nymphs in order to forecast the risk of TBEV for humans.