Recombinant proteins can be generated quickly and easily in large amounts and at low-cost in silkworm larvae by using Bombyx mori nuclear polyhedrosis virus (BmNPV). We searched for high-permissive silkworm strains that have high production levels of heterologous proteins and are thus suitable for use as biofactories. In this study, we performed the analysis using a BmNPV vector expressing luciferase as a marker, and we confirmed protein expression by evaluating luciferase activity, determined by western blotting and luciferase ELISA, and confirmed transcription expression by semi- and quantitative real time PCR. For the selection of host silkworm strains, we first chose 52 domestic BmNPV sensitive strains and then identified 10 high-permissive and 5 low-permissive strains. In addition, to determine which hybrid of the high-permissive strains would show heterosis, nine strains derived through three-way crossing were tested for luciferase activity by western blotting, and luciferase ELISA. We found a correlation between luciferase activity and luciferase protein expression, but not transcription. There was no noticeable difference in protein expression levels between Jam313 as the high-permissive control strain and the three-way hybrid strains; however, the three-way cross strains showed lower luciferase activity compared with Jam313. In this study, luciferase protein production in the larvae of 52 domestic silkworm strains was elucidated using BmNPV.