To investigate the effects of melatonin on the growth and physiological characteristics of hybrid Pennisetum urpureum ‘Guimu-1’ under drought stress, and to explore the physiological mechanism of melatonin alleviating the growth inhibition of hybrid P. urpureum ‘Guimu-1’ under drought stress, hybrid P. urpureum ‘Guimu-1’ plantlets under 10% PEG6000 drought stress were treated with six concentrations of melatonin solution by plant tissue culture. The differences in the responses of morphological and physiological indicators to different melatonin concentrations were analyzed. The results showed that drought stress significantly inhibited the growth of hybrid P. urpureum ‘Guimu-1’ plantlets, while adding different concentrations of melatonin could alleviate the growth inhibition caused by drought stress, and the most significant relief effect was observed when the melatonin concentration was 100 mg/L. Compared with the treatment of drought stress alone, the addition of 100 mg/L melatonin under drought stress increased the plant height, root length, stem length, leaf length, total fresh mass, aboveground fresh mass, underground fresh mass, and root shoot ratio of hybrid P. urpureum ‘Guimu-1’ plantlets by 74.4%, 69.9%, 110.9%, 75.4%, 97.6%, 80.5%, 118.1% and 20.2%, respectively; increased the relative water content, chlorophyll a, chlorophyll b, carotenoids, total root length, total root surface area, average root diameter, total root volume, root vitality, ascorbic acid (AsA) content, reduced glutathione (GSH) content, soluble sugar (SS) content, soluble protein (SP) content, proline content, CAT activity, SOD activity, POD activity, IAA content, GA3 content, ZR content, and ETH content by 40.2%, 69.2%, 117.3%, 141.3%, 87.8%, 38.3%, 62.5%, 35.9%, 59.2%, 113.6%, 80.8%, 72.2%, 52.5%, 25.7%, 48.7%, 82.5%, 27.5%, 38.5%, 49.8%, 55.4% and 9.1%, respectively; decreased the relative conductivity, ABA content, O2- content, H2O2 content and MDA content by 52.9%, 14.2%, 38.4%, 67.7%, 50.5%, respectively. Plant height, root length, stem length, leaf length, total fresh mass, aboveground fresh mass, underground fresh mass, root shoot ratio had significant or extremely significant positive correlations with relative water content, chlorophyll a, chlorophyll b, carotenoids, total root length, total root surface area, total root volume, root vitality, AsA, GSH, SS, SP, Pro, CAT, SOD, POD, IAA, GA3, ZR, ETH, and had significant or extremely significant negative correlations with relative conductivity, H2O2, ABA. Therefore, melatonin could significantly promote the growth of hybrid P. urpureum ‘Guimu-1’ plantlets under drought stress, enhance their antioxidant capacity, increase their osmoregulatory substance content, inhibit their excessive production of reactive oxygen species, and reduce their membrane peroxidation level, thereby improve the drought tolerance of hybrid P. urpureum ‘Guimu-1’ plantlets.