This study compared dexmedetomidine or xylazine in combination with tiletamine-zolazepam for chemical immobilization of wild fallow deer (Dama dama) in a prospective, randomized, blinded clinical study. Forty fallow deer were divided into two groups: tiletamine-zolazepam-xylazine (TZX) and tiletamine-zolazepam-dexmedetomidine (TZD). The TZX group was immobilized with 1.9 ± 0.05 mg/kg of xylazine and 1.48 ± 0.05 mg/kg of tiletamine-zolazepam, whereas the TZD group was immobilized with 34.15 ± 1.1 µg/kg of dexmedetomidine and 0.97 ± 0.03 mg/kg of tiletamine-zolazepam by dart. The induction time was recorded. During the immobilization, heart rate, respiratory rate, body temperature, hemoglobin oxygen saturation, blood lactate concentration, and quality of immobilization were recorded at 10, 20, and 30 m after drug administration. The time of achievement of sternal recumbency and that of standing were also recorded. The TZD group showed a significantly shorter induction time (8 ± 1.6 m, TZX group; 4 ± 0.5 m, TZD group), significantly higher quality of immobilization score (2[1-2], TZX group; 4[4-4], TZD group), and significantly lower lactate levels (5[3-7] mmol/L, TZX group; 2[1-3] mmol/L, TZD group). The time to sternal recumbency was 7 ± 1.6 m (TZX group) and 4 ± 0.5 m (TZD group), and time to quadrupedal standing was 20 ± 1.6 m (TZX group) and 16 ± 0.8 m (TZD group) (P = 0.001). Dexmedetomidine combined with tiletamine-zolazepam is a viable alternative to xylazine for the chemical immobilization of fallow deer.