The hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes two uracil DNA glycosylases (UDGs): Tba UDG247 and Tba UDG194. Herein, we characterized biochemically Tba UDG194. Compared with Tba UDG247, Tba UDG194 exhibits different biochemical characteristics. At >85 °C, >90 cleavage percentage was observed, suggesting that Tba UDG194 can remove uracil from DNA at physiological temperature of its host. Thus, the enzyme is the most thermophilic glycosylase among all the reported UDGs. Furthermore, the optimal pH of the enzyme activity was estimated to be 10, which is higher than that of Tba UDG247. Similar to Tba UDG247, Tba UDG194 activity is independent on a divalent metal ion. Mn2+, Zn2+ and Cu2+ display inhibitory effect on the enzyme activity at varied degreed whereas Mg2+ and Ca2+ have no detectable effect on the enzyme activity. In addition, Tba UDG194 is a salt-tolerant enzyme that retains compromised activity at 600 mM NaCl. Furthermore, Tba UDG 194 displays the following substrate preference: U ≈ U/G > U/T > U/A > U/C. The Arrhenius activation energy was estimated to be 20.1 ± 3.4 kcal/mol, theoretically representing the energy barrier for uracil removal from DNA by Tba UDG194. Overall, our observations suggest that Tba UDG194 might be involved in removal of uracil in DNA in Thermococcus cells.