This study aimed to evaluate the biochemical impact of magnesium (Mg) deficiency on banana cultivars. Seedlings of three banana cultivars, Baxi (Musa, AAA), Haigong (Musa, AA), and Guangfen no.1 (Musa, ABB), with five expanded leaves were sand cultured with nutrient solution containing 10 µM (Mg-deficient) and 1000 µM of MgSO4. After 12 weeks of Mg deficiency, Mg content in leaves and plant dry biomass was significantly reduced. The superoxide dismutase activity was increased in all three cultivars, while the photosynthesis seems to be unaffected. Differential responses were observed in different cultivars under Mg deficiency, and deficiency symptoms appeared on the leaves of Baxi and Haigong, while no symptoms appeared on Guangfen no. 1. Similarly, the increase in potassium, calcium, sucrose, starch contents and higher lipid peroxidation under Mg deficiency was observed in Baxi and Haigong, while unaffected in Guangfen no. 1. Also, antioxidant enzyme activity of ascorbate peroxidase and glutathione reductase was significantly increased in Haigong and Baxi, respectively. Mg deficiency reduced the biomass and root and shoot dry weight ratio, accompanied by the accumulation of malondialdehyde, sucrose, and starch content. The genetic differences are responsible for the tolerance of Mg deficiency in banana seedlings.