Abstract: Abstract: In this study, the antimicrobial crude extract was obtained as immunizing hemolymph from the last stage larvae of Calleria mellonella after induction. With three disparate treatment, fresh, frozen and heated, the crudes was carried out detection to two common bacteria and 16 kinds of plant pathogenic fungi, and separation with Sephadex G-50 gel filtration. The results showed that the three disparate treated substances have no visible antibacterial effect to E. coli , but a obvious effect to Staphylococcus aureus with inhibition of about 10 bacteriostasis units; have dissimilar effects to 10 kinds of fungi among the total kinds, and have a good inhibitory effect to Cytospora.coccodes (Wallr.)Hughes with fungistasis rate above 65%. After the gel filtration and activity detection, 50mM ammonium acetate was screened out as the more suitable eluents, the crudes were separated into strikingly unlike parts with obviously different antimicrobial activity.