This investigation was conducted in the tissue culture laboratory vegetable department, Faculty of Agriculture, AssiutUniversity, to study the effect of Magnesium Sulphate (MgSO₄) and light duration in-vitro multiplication rate of five subculture on banana. Magnesium Sulphate (MgSO₄) was add at 1, 2.5, 5 and 10 ppm and light duration treatment applied at (darkness for 4 weeks, natural light for 4 weeks, 16 hours light for 4 weeks and 2 weeks at darkness followed by 2 weeks at 16 hours light). Results indicated that light duration treatment are more significantly effect on multiplication rate, chlorophyll a, chlorophyll b and carotene contents. Darkness for 2 weeks followed by light 16 hours for 2 weeks recorded the highest rate of multiplication rate. Meanwhile, Magnesium Sulphate and their interactions with light duration treatments are of non-significant effect on multiplication rate. The best results was regard to multiplication and improvement of plant morphogenesis at final stage of development in vitro of Grand Naine Banana when explants incubated at  dark for a period 15 days followed by  16 hours light for 15 days.