Given the significant differences in amino acid composition among proteins from various sources, the development of aminopeptidases with broad substrate spectrum can effectively support the efficient and directional hydrolysis of proteins. To overcome the narrow substrate spectrum of existing aminopeptidases, a novel aminopeptidase gene APs(Ar)-3 was screened and identified from Acinetobacter radioresistens a2, which was heterologously expressed in Escherichia coli. Enzymatic properties of APs(Ar)-3 were identified, and it was applied to the hydrolysis of oyster proteins. The results showed that APs(Ar)-3 exhibited a broad substrate spectrum, displaying prominent catalytic activity towards Ala-pNA and Arg-pNA, and was also capable of hydrolyzing hydrophobic amino acids such as Met and Leu. The optimal reaction temperature was 45℃, and the optimal reaction pH was 7.0. The enzyme maintained good stability at temperatures below 45℃ and within the pH range of 6.0-8.0. Co²⁺ at 0.1mmol/L could significantly activate the aminopeptidase activity of APs(Ar)-3, while Zn²⁺ and Cu²⁺ had inhibitory effects on the enzyme. In the enzymatic hydrolysis of oyster proteins, the degree of hydrolysis achieved by the synergistic action of APs(Ar)-3 with bromelain and trypsin were 57.86% and 57.61%, respectively, which were 17.2% and 10.69% higher than those of commercial aminopeptidases. Moreover, the addition of APs(Ar)-3 increased the umami taste value of the oyster protein hydrolysate and reduced its bitterness and astringency. This study aimed to provide an efficient aminopeptidase with a broad substrate spectrum for the preparation of protein hydrolysates, and offer theoretical reference and technical support for the directional hydrolysis of proteins and the high-value development and application of oyster resources.