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Insects have evolved to form a variety of complex natural compounds to prevent pathogen infection in the process of a long-term attack and defense game with various pathogens in nature. Antimicrobial Peptides (AMPs) are important effector molecules of the insect immune response to the pathogen invasion involved in bacteria, fungi, viruses and nematodes. The discovery and creation of new nematicides from these natural compounds is a key path to pest control. A total of 11 AMPs from <i>Monochamus alternatus</i> were classified into 3 categories, including Attacin, Cecropin and Defensin. Four AMP genes were successfully expressed by <i>Komagataella phaffii</i> KM71. The bioassay results showed that the exogenous expressed AMPs represented antimicrobial activity against <i>Serratia</i> (G⁻), <i>Bacillus thuringiensis</i> (G⁺) and <i>Beauveria bassiana</i> and high nematicide activity against <i>Bursaphelenchus xylophilus</i>. All four purified AMPs’ protein against <i>B. xylophilus</i> reached LC₅₀ at 3 h (LC₅₀ = 0.19 mg·mL⁻¹ of MaltAtt-1, LC₅₀ = 0.20 mg·mL⁻¹ of MaltAtt-2 and MaltCec-2, LC₅₀ = 0.25 mg·mL⁻¹ of MaltDef-1). Furthermore, the AMPs could cause significant reduction of the thrashing frequency and egg hatching rate, and the deformation or fracture of the body wall of <i>B. xylophilus.</i> Therefore, this study is a foundation for further study of insect biological control and provides a theoretical basis for the research and development of new insecticidal pesticides.