Bombyx mori is an important economic insect and a classic Lepidoptera model insect. Although the immune-related silkworm genes have been identified on a genome-wide scale in the silkworm, the immune defense of silkworm in protein level was still unclear. Fat body synthesizes immune-related proteins and secreted them into hemolymph when pathogens invade in B.mori. In this study, two types of bacteria were injected into the silkworm, respectively, including the gram-negative Escherichia coli and gram-positive Staphylococcus aureus. Proteomic analyses of the hemolymph proteins after injection were performed by LC-MS/MS. A total of 514 proteins were identified in the uninduced group, 540 were identified in the E.coli inducted group, and 537 were identified in the S.aureus induced group. Based on the uniprot annotation, this study identified 32 immunological recognition proteins, 28 immunological signaling proteins, and 21 immunological effectors. We found that 127 proteins showed significant up-regulation, including 10 immunological recognition proteins, 4 immunological signaling proteins, 11 immunological effector proteins, and 102 other proteins. The qRT-PCR was then employed to verify whether the up-regulated proteins were also response to bacteria at the transcription level. As expected, immunological recognition proteins, signaling proteins, and effector proteins showed a significant increase at the transcriptional level after being infected with E.coli and S.aureus. Some other proteins also showed up-regulation after infection, such as 30K protein, yellow-d protein, chemosensory protein, and uncharacterized proteins. This research screened many new immune-related proteins and deepened our understanding on immune defense system in B.mori.