A thermostable $eta$-glycosidase gene, tfi $eta$-gly, was cloned from the genomic library of Thermus filiformis Wai33 A1. ifi $eta$-gly consists of 1,296 bp nucleotide sequence and encodes a polypeptide of 431 amino acids. It shares a strong amino acid sequence similarity with the $eta$-glycosidases from other Thermus spp. belonging to the glycosyl hydrolase family 1. In the present study, the enzyme was overexpressed in Escherichia coli BL21 (DE3) using the pET21b(+) vector system. The recombinant enzyme was purified to homogeneity by heat treatment and a $Ni^{2+}$-affinity chromatography. Polyacrylamide gel electrophoresis (PAGE) showed that the recombinant Tfi $eta$-glycosidase was a monomeric form with molecular mass of 49 kDa. The temperature and pH range for optimal activity of the purified enzyme were 80- $90^{circ}C$ and 5.0-6.0, respectively. Ninety-three percent of the enzyme activity was remained at $70^{circ}C$ after 12 h, and its half-life at $80^{circ}C$ was 6 h, indicating that Tfi $eta$-glycosidase is highly thermostable. Based on its K_m$, or $K_{cat}K_m$, ratio, Tfi $eta$-glycosidase appeared to have higher affinity for $eta$-D-glucoside than for $eta$-D-galactoside, however, $K_{cat} for eta$-D-galactoside was much higher than that for $eta$-D-glucoside. The activity for lactose hydrolysis was proportionally increased at $70^{circ}C$ and pH 7.0 without substrate inhibition until reaching 250 mM lactose concentration. The specific activity of Tfi TEX>$eta$</TEX>-glycosidase on 138 mM lactose at $70{^circ}C$ and pH 7.0 was 134.9 U/mg. Consequently, this newly cloned enzyme appears to have a valuable advantage of conducting biotechnological processes at elevated temperature during milk pasteurization in the production of low-lactose milk.