This study was conducted with Yongyou 1540 and Huazheyou 261 as experimental materials to explore the effects of seedling cultivation using rock wool and a specialized substrate for rice seedling-raising on seedling growth, physiological characteristics, and rice yield under different moisture conditions. The results showed that in terms of emergence rate, the specialized substrate for rice seedling-raising performed better than rock wool seedling-raising. However, under conditions of high temperature and water stress, seedlings raised with rock wool exhibited advantages over those raised with the specialized substrate in terms of plant height and dry matter accumulation. Moreover, rock wool seedling-raising also increased the soluble sugar content and elevated the carbon-to-nitrogen ratio in the seedlings. Additionally, compared to the specialized substrate for rice seedling-raising, rock wool had better water retention properties, which effectively enhanced the drought resistance of rice seedlings. In terms of yield, rock wool seedling-raising also demonstrated certain advantages, showing an 8.2% increase compared to seedling-raising with the specialized substrate.