Aminopeptidase is a kind of exopeptidases that specifically cleaves amino acids from the N-terminal of proteins, and thus has wild applications in food industry. In this study, an aminopeptidase was isolated to homogeneity from oysters (Alectryonella plicatula) by ammonium sulfate fractionation and a series of chromatographic steps including DEAE-sepharose, phenyl-sepharose and hydroxyapatite. The aminopeptidase was strongly suppressed by bestatin, a specific aminopeptidase inhibitor. The 2D-PAGE results showed that molecular weight and pI value was approximately 100 ku and 5.8. By MALDI-TOF/TOF mass spectrometry analysis, 12 peptide fragments with 138 amino acid residues in total were obtained and these peptide fragments were identical to a puromycin-sensitive aminopeptidase from Crassostrea gigas, which confirmed that the purified enzyme was an aminopeptidase B. Circular dichroism spectroscopy analysis indicated that the secondary structure of aminopeptidase B (APB) was mainly random coil (42.6%) and reverse parallel (32.0%). The K_m and k_cat was 1.5μmol/L and 117.5s-1 and the k_cat/K_m was 78.3L/(μmol·s)-1. The optimum temperature and pH of APB was 35℃ and 7.0. Under these conditions, APB could hydrolyze Lys-MCA and Arg-MCA to release Lys and Arg, which had the oyster’s flavor.