Acetylcholinesterase (AChE) has emerged as a significant biological recognition element in the biosensor field, particularly for the detection of insecticides. Nevertheless, the weak thermostability of AChE restricts its utilization due to the complexities associated with production, storage, and application environments. By evaluating the binding affinity between representative AChE and insecticides, an AChE from Culex pipiens was screened out, which displayed a broad-spectrum and high sensitivity to insecticides. The C. pipiens AChE (CpA) was subsequently expressed in Escherichia coli (E. coli) as a soluble active protein. Furthermore, a three-point mutant, M4 (A340P/D390E/S581P), was obtained using a semi-rational design strategy that combined molecular dynamics (MD) simulation and computer-aided design, which exhibited a four-fold increase in half-life at 40 °C compared to the wild-type (WT) enzyme. The mutant M4 also demonstrated an optimal temperature of 50 °C and a melting temperature (T) of 51.2 °C. Additionally, the sensitivity of WT and M4 to acephate was examined, revealing a 50-fold decrease in the IC value of M4. The mechanism underlying the improvement in thermal performance was elucidated through secondary structure analysis and MD simulations, indicating an increase in the proportion of protein helices and local structural rigidity. MD analysis of the protein-ligand complexes suggested that the enhanced sensitivity of M4 could be attributed to frequent specific contacts between the organophosphorus (OP) group of acephate and the key active site residue Ser327. These findings have expanded the possibilities for the development of more reliable and effective industrial enzyme preparations and biosensors.