Roe deer Capreolus capreolus are among the most important feeding hosts for the sheep tick Ixodes ricinus, thus contributing to the occurrence of tick-borne diseases in Europe. Tick-borne encephalitis (TBE), which is transmitted by co-feeding of larvae and nymphs on rodents, requires precise climatic conditions to occur. We used roe deer as sentinels for potential circulation of TBE virus in Northern Italy, by examining the association between tick infestation, occurrence of TBE human cases, geographical and climatic parameters. Tick infestation on roe deer, and particularly frequency of co-feeding, was clearly associated with the geographic location and the autumnal cooling rate. Consistently, TBE occurrence in humans was geographically related to co-feeding tick abundance. The surveillance of tick infestation on roe deer, combined with remotely sensed climatic data, could therefore be used as an inexpensive early risk assessment tool of favourable conditions for TBE emergence and persistence in humans.