Tick-borne encephalitis (TBE) is a serious and acute central nervous system infection caused by the tick-borne encephalitis virus (TBEV). In recent years, TBE has emerged as a growing public health threat, with cases reported across Europe, the Russian Far East, Japan and China. This study aims to assess the prevalence of TBEV infection and examine behaviours associated with an increased risk of infection among individuals who visited the Heilongjiang Red Cross Sengong General Hospital due to tick bites from 2020 to 2023. We collected blood samples and administered survey questionnaires from tick-bitten people. A total of 457 samples were screened using Nested PCR, and the detected TBEV prevalence rate was 29.54% (135/457). The symptoms of redness and swelling at the site of tick bite (42.57%), fever (28.71%) and headache (10.89%) were identified in the TBEV-positive individuals when they visited the hospital by the physician. Phylogenetic analysis of the partial E gene of TBEV revealed that the predominant strains in the region are highly virulent Far Eastern subtype. However, they do not cluster with the three established evolutionary clades of the Far Eastern type. Questionnaires data analysis identified age and first tick bite as important factors associated with TBEV infection. This study provides basic information on the epidemiology of TBEV in Heilongjiang Province in recent years and identifies that the most related risk factor of infecting TBEV is tick exposure. Further research is needed to develop effective prevention and control measures.