The objective of this study was to screen and clone a leucine aminopeptidase gene (EsLAP) from the transcriptome of Euphausia superba, construct the recombinant expression vector EsLAP-PET-28A, and achieve soluble expression in Escherichia coli through co-expression of a molecular chaperone. The induction conditions were optimized by using the three-factor and three-level response surface method, and the enzymatic properties of the recombinant enzyme were systematically characterized. The total length of the EsLAP gene was 1569 bp, and it encoded 522 amino acids, with a theoretical molecular weight of 55302.67 Da and an isoelectric point of 6.16. Sequence and structural analysis indicated that EsLAP possesses typical peptidase catalytic domains and substrate-specific binding sites of M17 family peptidases. The optimal expression conditions of EsLAP were determined through response surface optimization as follows: IPTG concentration of 0.7 mmol/L, inoculation dose of 3%, and induction time of 19 h. Enzymatic property analysis showed that the optimal reaction temperature of EsLAP was 60 ℃, the optimal pH was 8.5, the crude enzyme activity was 265.6 U/mL, and the specific enzyme activity was 33.15 U/mg. Metal ions Co²⁺ and Mn²⁺ can significantly enhance enzyme activity, among which Co²⁺ can increase enzyme activity to 296% of the original level. EDTA can significantly inhibit its enzymatic activity, indicating that EsLAP is a metalloproteinase. Strong reducing agents such as β-mercaptoethanol, NaBH₄, and DTT significantly inhibit enzyme activity and can reduce the remaining enzyme activity to less than 10%. Substrate specificity analysis showed that EsLAP had the highest catalytic efficiency for Leu-pNA, further verifying that it was leucine aminopeptidase. This is the first report on the heterologous expression and enzymatic characteristics of leucine aminopeptidase derived from Euphausia superba. The related results provide a potential application basis for its use in the food and pharmaceutical industries to degrade N-terminal leucine-containing functional peptides.