Abstract: To investigate the effects of different substrate compositions and plant growth regulators on the rooting and seedling growth of Melliodendron xylocarpum hardwood cuttings, a four-factor, three-level orthogonal experimental design was conducted. The study systematically analyzed the effects of substrate composition, type and concentration of growth regulators, and treatment duration on root morphology, and seedling growth. The results demonstrated that a vermiculite: perlite (3∶1) substrate promoted root diameter thickening but inhibited vertical growth, whereas a river sand: vermiculite (1∶1) mixture enhanced specific root length, shoot elongation, and biomass accumulation. Pure vermiculite significantly improved root number and surface area. Among growth regulators, IAA treatment effectively enhanced rooting percentage and specific root length. A concentration of 500 mg/L significantly increased rooting rate and aboveground biomass. A 30-minute treatment duration yielded the most favorable outcomes across multiple parameters. Range analysis indicated that growth regulator concentration exerted the greatest influence on overall performance, followed by treatment duration, substrate composition, and growth regulator type.