To identify the optimal fertilization formula for nutrient soil in rice seedling cultivation, the variety Nanjing 9108 was used as experimental material. Different fertilizer formulations were applied to prepare the nutrient soil. A total of 14 treatments were designed, including treatment 1 without adding fertilizer; treatment 2, the substrate ratio was seedling green∶bed bottom fertilizer∶soil=1∶0.5∶200; treatment 3, seedling green∶acid regulator∶soil=1∶0.15∶150; treatment 4-6, seedling green∶acid regulator∶soil=1∶0.3∶150; treatment 7, seedling green∶acid regulator∶soil=1∶0.45∶150; treatment 8, seedling green∶acid regulator∶bed bottom fertilizer∶soil=1∶0.3∶0.5∶150; treatment 9-13, seedling green∶acid regulator∶bed bottom fertilizer∶soil=1∶0.3∶0.5∶200; treatment 14, seedling green∶acid regulator∶bed bottom fertilizer∶soil=1∶0.3∶0.5∶250; among them, treatment 4 and 5 received base fertilizer at 27 g/tray, treatment 12, received diammonium phosphate (1.5 g/tray)+urea (1 g/tray) at 9 and 16 days after tray moving, treatment 13 received ammonium sulfate (3.5 g/tray) at the same intervals, other treatments received urea (2 g/tray) at the same intervals; treatment 9, seeds (9 kg) treated with 60 g mefenoxam·prochloraz, treatment 11 seeds (9 kg) treated with 60 g mefenoxam·prochloraz+0.3 g 15% paclobutrazol, other treatments: seeds (9 kg) treated with 60 g mefenoxam·prochloraz+0.15 g 15% paclobutrazol. The effects of different substrate formulations and fertilization management on seedling growth was investigated. The results showed that treatments 1, 5, 3, 7, 4, 11, 13, 6, and 14 exhibited higher emergence rates, ranging from 57.50% to 68.67%. Plant height across treatments varied between 12.44-20.52 cm, leaf age ranged from 4.86 to 5.53 leaves, white root number measured 11.80-14.37 roots per plant, stem base width spanned 2.00-2.70 mm, and SPAD values fell within 22.07-30.37. Comprehensive analysis of emergence rate, white root number, leaf color, and other parameters identified treatment 11 (seedling green∶acid regulator∶bed bottom fertilizer∶soil=1∶0.3∶0.5∶200; seed treatment, 60 g mefenoxam·prochloraz + 0.3 g 15% paclobutrazol per 9 kg seeds; urea application of 2 g/tray at 9 and 16 days after tray relocation) as optimal. This treatment demonstrated above average performance in emergence rate, white root number, leaf age, and stem base width, proving conducive to cultivating robust seedlings and suitable for production application.