D-Tagatose is a representative rare sugar with the physiochemical properties of low energy and high sweetness, as well as excellent physiological functions such as blood sugar regulation, enhancement of intestinal flora, and prevention of dental caries. At present, D-tagatose production involves lactose hydrolysis and D-galactose isomerization processes, resulting in high production costs that hinder its industrial advancement. Tagatose 4-epimerase (T4Ease) has the capability to directly convert d-fructose into D-tagatose through C-4 epimerization, providing a new approach for D-tagatose production. In this study, a hyperthermostable T4Ease from Infirmifilum uzonense (Inuz-TE4ase) was identified from the Foldseek clustered AlphaFold database and its biochemical properties were characterized in detail. Under the optimal reaction conditions of 90 °C and pH 8.5 (Tris-HCl) with the addition of 1 mM Ni