Defense peptides and proteins constitute key factors in insect humoral immune response against invading microorganisms. In this study, biochemical approach was designed to purify and characterize two peptides which appeared in larval haemolymph of B. mori after bacterial challenge. The results showed a significant increase of total protein of the bacterial-challenged haemolymph and then declined over time. This suggested that the AMPs are upregulated and released in haemolymph as “acute phase response" of the insect. Full antimicrobial activity was observed for the immune haemolymph at 24 h p.i. Fractionation of the immune haemolymph extract on a reversed phase C-18 column allowed effective separation of 5 fractions containing mainly proteins and peptides of molecular masses below 20 kDa. After fractionation, one out of three fractions (fraction# 5) exhibited the strongest antimicrobial activity. Finally, two peptides (5.8 and 4.3 KDa) were purified and one of them (4.3 KDa) showed full antimicrobial activity and very weak hemolytic activity up to concentration of 100 µg/ ml. These results were consistent to the results of quantitative protein analysis. Conclusively, this study demonstrated that the antimicrobial activity of the immune haemplymph is related to the presence of two antimicrobial peptides.