Abstract: L-Asparaginase (ASNase), an aminohydrolase, is widely utilized in the pharmaceutical and food industries. Among its various sources, Escherichia coli K12-derived EcASNase has been employed as a clinical drug for the treatment of acute lymphoblastic leukemia (ALL). However, the limited catalytic activity and stability of EcASNase have restricted its broader application in medicine and food processing. In this study, a random mutagenesis library was constructed via error-prone PCR, followed by high-throughput screening using a coupled bacterial growth strategy. Three positive mutants with enhanced activity were identified: G38S, Q212Y, and S274P, exhibiting activities 1.4-, 1.1-, and 1.2-fold higher than the wild type (WT), respectively. Saturation mutagenesis libraries were subsequently generated for positions 38, 212, and 274, leading to the identification of mutants G38A, G38S, G38Q and G38V, with k_cat/K_m values 1.7-, 1.5-, 2.1-, and 2.2-fold higher than WT, respectively. Among these, G38V emerged as the most active mutant, with a T_m value increased by 8.4 ℃ compared to WT. Combination mutations, such as G38V/Q212F and G38V/S274P, failed to yield further activity improvements. This research elucidates the contributions of critical residues to the enzyme's activity and stability, providing novel insights into the rational design and development of therapeutic enzymes.