The melatonin biosynthesis in plant is mainly catalyzed by tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), serotonin N-acetyltransferase (SNAT) and N-acetylserotonin methyltransferase (ASMT). In this study, nine MaTDCs, one MaT5H, one MaSNAT, and twenty-four MaASMTs were identified from Musa acuminate genome. The MaTDC and MaASMT gene families were further studied through bioinformatics analysis and expression pattern analysis under low temperature and exogenous melatonin treatments. MaTDCs and MaASMTs could be classified into two and five subfamilies, respectively. These classifications were strongly supported by their gene structures and protein conserved motifs. The expression of MaTDC4–1, MaTDC7–1, MaTDC8–2, MaTDC10–1 and MaASMT10–1 and the content of endogenous melatonin in banana leaf were significantly promoted by low temperature. Moreover, the promoting effects of combined low temperature and exogenous melatonin treatment were more significant. Notably, the expression of MaTDC4–1, MaTDC8–2, MaTDC10–1 and MaASMT10–1 was not only low temperature inducible but also very significantly or significantly positively correlated with the melatonin content. Moreover, transient overexpression of MaTDC4–1 and MaASMT4–2 greatly increased the melatonin accumulation in tobacco leaf. Our study revealed the contributions of MaTDC and MaASMT genes and melatonin to the banana cold resistance.