The gene sequence of acetylcholinesterase was optimized based on codon versatility and the principle of difference codon usage frequency of different species， aiming to increase its expression in Pichia pastoris and enhance its application prospects. The low frequency codons in the gene sequence were replaced with Pichia pastoris preferred codons， while the GC content of each segment of the acetylcholinesterase was adjusted to 45%-55%. The optimized gene was ligated into the expression vector pPIC9K and transferred into Pichia pastoris GS115， and cultured in shake flasks. The enzymatic properties of the recombinant enzyme mdAchE were determined by the modified Ellman method， and the bioactivity of the recombinant enzyme mdAchE was analyzed by organophosphorus and carbamate pesticide inhibition assays. The mdAch E codon was optimized to achieve high expression in Pichia pastoris with an enzyme activity of approximately 8.82 U/mL. The inhibition test showed that the acetylcholinesterase could be inhibited by seven organophosphorus pesticides such as malathion and eight carbamate pesticides such as carbofuran. The minimum inhibitory concentration IC15 value was 1.83×10-15 μg/mL， which was far lower than the lowest detection limit （0.01 μg/mL， 4.14×10-6 μg/mL） measured by other methods such as gas chromatography， biosensors. The recombinant enzyme mdAchE has great application prospects in the detection of organophosphorus and carbamate pesticide residues.