Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 is the key membrane fusion protein that mediates budded virus (BV) infection. We recently reported that BmNPV GP64's n-region of signal peptide (SP) blocked the SP-cleavage and mediated GP64 localization on the plasma membrane (PM); n-region (SP ∆n GP64) absence caused GP64 intracellular localization, however, SP ∆n GP64 was still incorporated into virion to generate BVs with lower infectivity. To better understand the biogenesis of the envelope of BmNPV BV, we conducted a label-free ESI mass spectrometry analysis of the envelope of purified BVs harboring PM localized GP64 or intracellular localized SP ∆n GP64. The results indicated that 31 viral proteins were identified on the envelope, among which 15 were reported in other viruses. The other 16 proteins were first reported in BmNPV BV, including the BmNPV-specific protein BRO-A and proteins associated with vesicle transportation. Six proteins with significant intensity differences were detected in virions with differential localized GP64, and five specific proteins were identified in virions with GP64. Meanwhile, we identified 81 host proteins on the envelope, and seven lipoproteins were first identified in baculovirus virion; other 74 proteins are involved in the cytoskeleton, DNA-binding, vesicle transport, etc. In the meantime, eight and five specific host proteins were, respectively, identified in GP64 and SP ∆n GP64's virions. The two virions shared 68 common host proteins, and 8 proteins were identified on their envelopes with a significant difference. This study provides new insight into the protein composition of BmNPV BV and a clue for further investigation of the budding mechanism of BmNPV.