This study constructed a full-length infectious clone of tick-borne encephalitis virus (TBEV), providing an essential tool for in-depth investigation of TBEV replication and pathogenesis. Firstly, prokaryotic promoters within the full-length TBEV sequence were predicted, and synonymous mutations were introduced to eliminate the activity of these promoters, thereby reducing the bacterial toxicity of the cDNA. Subsequently, considering sequence complexity and cloning efficiency, the TBEV cDNA sequence was divided into two fragments for synthesis, which were then cloned into the pFK vector using homologous recombination technology. Viral RNA generated by in vitro transcription was transfected into cells. Viral RNA replication was successfully detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) using TBEV E protein-specific primers. Meanwhile, Western blot analysis with a TBEV NS1 protein-specific antibody confirmed viral protein expression. 50% tissue culture infectious dose (TCID50) assays demonstrated a significant increase in viral titers at 48 h post-transfection. Finally, plaque assays in BHK-21 cells at 96 h post-infection validated the infectivity of second-generation viruses. Results showed that plaque numbers markedly increased with the decreasing viral dilution, and both viral RNA levels and protein expression escalated over time, indicating the infectivity and intracellular replication capacity of second-generation viruses. These results provide a novel tool for TBEV research, and a general strategy for constructing infectious clones.