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Acyl-peptide releasing enzyme(AARE) belongs to a serine peptidase family and catalyzes the NH2-terminal hydrolysis of Nα-acylpeptides to release Nα-acylated amino acids. ORF0779(ORF=open reading frame) from thermophilic archaea Sulfolobus tokodaii(ST0779) was cloned and expressed in E. coli BL21 and the expressed protein was identified as a thermostable AARE. The target protein could be optimally overexpressed in E. coli at 30 °C for 8 h with 0.1 mmol/L isopropyl β-dthiogalactoside(IPTG). The crude enzyme was heated at 70 °C for 30 min, and then the target protein could account for above 40% of the total protein. The purification fold was 27 and the enzyme showed both esterase activity and peptidase activity. The optimal temperature and pH for ST0779 were 70 °C and 8.0 when Ac-Ala3 was used as substrate. The half-life of the enzyme(0.2 mg/mL) at 90 °C was about 16 h, indicating that the enzyme exhibits a favorable thermostability. The activity of ST0779 could still remain over 85% after being treated at 25 °C in different buffers with pH range from 6.0 to10.0 for 24 h, which indicates ST0779 is stable in neutral or slight alkali environment. Under neutral or slightly alkali conditions, the enzyme exhibits really high catalytic efficiency against acyl-peptide, and the optimal substrate is Ac-Ala3. Most metal ions have no inhibition effect on the activity of ST0779, while 4% activity of ST0779 is inhibited in the presence of K+. This enzyme was supposed to be applied in the analysis of protein sequencing and the synthesis of small peptides.