Aims The interplay between melatonin and brassinosteroids in enhancing tolerance to cold and drought and the underlying molecular mechanisms of this relationship still remain unknown. Methods Combined physiological and transcriptomic analyses were used to clarify the crosstalk of melatonin and brassinosteroids in perennial ryegrass. Brassinosteroids biosynthesis inhibitor propiconazole, Arabidopsis brassinosteroids-receptor mutant bri1-9, bak1, and H2O2 deficient rbohC and rbohF mutants were used to analyzed the role of H2O2 in melatonin and brassinosteroids-mediated tolerance to the stress. Results Melatonin-enhanced cold and drought tolerance of perennial ryegrass depends on the duration of dose and stress that were applied. Melatonin activates the expression of NADPH oxidase-related genes to promote H2O2 production when exposed to short-term cold/drought stresses but reduced accumulation of H2O2 under long-term stress. This was associated with an increase in enzymatic and non-enzymatic antioxidant activities by regulation of their gene expression and modulation of the ascorbate-glutathione cycle. Moreover, exogenous melatonin increased the biosynthesis of melatonin and brassinosteroids or the expression of signaling-related genes. However, the effects of melatonin on the expression of BR biosynthesis and signaling-related genes were inhibited in the rbohC and rbohF mutants. Chemical scavenging of H2O2 attenuated melatonin-mediated growth in the Arabidopsis wild-type, but the bak1 and bri1.9 mutants were relatively insensitive. Consistently, Inhibition of H2O2 production impaired the effect of melatonin and brassinosteroids on seed germination and the root growth of perennial ryegrass. Conclusions This research reveals a novel regulatory mechanism of the crosstalk of H2O2 and brassinosteroid signaling in melatonin-induced cold/drought tolerance in perennial ryegrass.