Mango (Mangifera indica L.) is one of the most significant tropical and subtropical fruit species, with high ecological and economic value. However, research on the in vitro culture of mangoes is relatively weak, so establishing an efficient and stable mango plant regeneration system is of great significance. In this study, a preliminary mango regeneration system was established with Mangifera indica L. cv. Keitt from young branches as the starting explants. The results showed that the optimal plant growth regulator (PGR) formula for direct adventitious shoot induction on the branches was 1 mg/L 6-benzylaminopurine (6-BA) + 0.1 mg/L a-naphthaleneacetic acid (NAA), with an adventitious shoot induction rate of 73.63% and an average of 6.76 adventitious shoots. The optimal basal medium for adventitious shoot induction was wood plant medium (WPM), with an adventitious shoot induction rate of 63.87% and an average of 5.21 adventitious shoots. The optimal culture medium for adventitious shoot elongation was WPM + 1 mg/L 6-BA + 0.5 mg/L NAA, with an adventitious shoot elongation rate of 89.33% and an average length of 5.17 cm. The optimal formula for the induction of mango rooting was Douglas fir cotyledon revised medium (DCR) + 3 mg/L indole-3-butyric acid (IBA), with a maximum rooting rate of 66.13% and an average rooting quantity of 6.43. The genetic fidelity of the in vitro-regenerated plants was evaluated using inter-simple sequence repeat (ISSR) molecular markers. There was no difference between the in vitro-regenerated plants and the parent plant. This study provides an efficient and stable propagation system for Mangifera indica L., laying the foundation for its rapid propagation and genetic improvement.