Abstract: PR10 is an important member of the pathogenesis-related protein (PR) family, which enhances the ability of plants to resist external stresses. In this study, a PR10 gene from Picea asperata Mast. PaPR10-1 was obtained by RT-PCR, with sequence characteristics then analyzed and prokaryotic expression system further constructed and optimized. Ribonuclease activity was then analyzed using the substrate method in vitro. Results showed that the open reading frame (ORF) of PaPR10-1 was 486 bp in length and encoded a protein with a 161 amino acid. Without a transmembrane structure and signal peptide, the PaPR10-1 protein was an intracellular protein with two conserved domains, “P-Loop” and Bet-v1-like, respectively. Phylogenetic analysis showed that PaPR10-1 was closely related to the PR10 protein of Pinus pinaster Aiton. Optimal expression conditions for the gene were 0.2 mmol/L IPTG at 30℃ for 1 h induction, and the expressed target protein exhibited ribonuclease activity.