A novel ginsenoside-hydrolyzing ${eta}$-glucosidase was purified from Paecilomyces Bainier sp. 229 by a combination of Q-Sepharose FF, phenyl-Sepharose CL-4B, and CHT ceramic hydroxyapatite column chromatography. The purified enzyme was a monomeric protein with a molecular mass estimated to be 115 kDa. The optimal enzyme activity was observed at pH 3.5 and $60^{circ}C$. It was highly stable within pH 3-9 and at temperatures lower than $55^{circ}C$. The enzyme was specific to ${eta}$-glucoside. The order of enzyme activities against different types of ${eta}$-glucosidic linkages was ${eta}$-(1-6)>${eta}$-(1-2)>${eta}$-(1-4). The enzyme converted ginsenoside Rb1 to CK specifically and efficiently. An 84.3% amount of ginsenoside Rb1, with an initial concentration of 2 mM, was converted into CK in 24 h by the enzyme at $45^{circ}C$ and pH 3.5. The hydrolysis pathway of ginsenoside Rb1 by the enzyme was $Rb1{ o}Rd{ o}F2{ o}CK$. Five tryptic peptide fragments of the enzyme were identified by a newly developed de novo sequencing method of post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. By comparing the five identified peptide sequences with the NCBI database, this purified ${eta}$-glucosidase proves to be a new protein that has not been reported before.