[Aim] Bt protein spatial-temporal expression and insect-resistance to three Lepidoptera pests, Ostrinia furnacalis, Mythimna separata, and Helicoverpa armigera, were studied in order to provide scientific data for the commercialization of transgenic cry1Ab/cry2Aj and G10ev-epsps maize double resistant 505-12-5.[Method] Spatial-temporal expression patterns of Bt protein were studied by ELISA. Field and laboratory insect resistance was tested by artificial grafting and in vitro tissue bioassay.[Result] At different developmental stages, the content of Bt proteins significantly varied in the different organs. At 6-8 leaf stage, Bt protein contents were:root (517.3 ng·g^-1) > leaf (453.8 ng·g^-1) > stem (312.8 ng·g^-1). At the bell stage, Bt protein contents were:leaf (353.3 ng·g^-1) > root (281.3 ng·g^-1) > stem (232.9 ng·g^-1). At the silking stage, Bt protein contents were:leaf (188.9 ng·g^-1) > root (114.1 ng·g^-1) > stem (53.6 ng·g^-1). At the milky stage, Bt protein contents were similar in three organs, root (178.0 ng·g^-1), stem (160.3 ng·g^-1) and leaf (185.4 ng·g^-1). Bt protein contents in reproductive organs were:seed (181.3 ng·g^-1)> filament (100.1 ng·g^-1)> pollen (95.0 ng·g^-1)> tassel (79.8 ng·g^-1). The results of laboratory insect-resistant test showed that armyworm mortality rates were low after 24 h feeding with young leaves of 505-12-5, and reached to 98.21% mortality after 48 h of feeding. The corn borer larval mortality rates were 87.37%, 100% and 100%, respectively, after 24 h feeding with young leaves, filaments, and seeds of 505-12-5. The mortality rate of cotton bollworm was 80.18% after 24 h of feeding with filaments of 505-12-5 and 92.45% after 48 h. Insect resistance tests in the field also showed that 505-12-5 was highly resistant to corn borer in whorl stage and female ear stage, to armyworm in whorl stage, and to cotton bollworm at silking stage.[Conclusion] Bt protein expressed in different organs of transgenic maize double resistance 505-12-5 at different growth stages. The expression of Bt protein was higher in pest susceptible organs at 6-8 leaf, silking and milky stages. Double resistant 505-12-5 transgenic maize has the potential for application because of its significantly high resistance to three Lepidoptera pests in the field and laboratory.