Abstract: Five hundred and forty five-month-old seedlings of Eucalyptus maidenii were employed to elucidate the mechanism of fertilization regulation via the regression-orthogonal experimental design of two factors (i.e., N and P) and three levels. The effects of fertilization on the growth, biomass accumulation, and nutrient physiology of the seedlings were analyzed. The optimal amount of fertilizer (i.e., N and P) for the seedlings was stated. The results showed that the optimal experimental treatment for the growth of seedling height and ground diameter was the fifth experimental treatment (N fertilizer 0.55 g/plant and P fertilizer 0.60 g/plant). The promotion effects of N fertilizer on the growth of seedling height and the accumulation of total biomass were better than that of P fertilizer, and the promotion effect of P fertilizer on the growth of ground diameter was better than that of N fertilizer. The growth of seedling height and ground diameter, the accumulation of total biomass all first increased and then decreases with the higher content of N and P fertilizer in the seedling substrate. The promotion effects of N and P fertilizer combination on the growth of seedling height, ground diameter and the accumulation of total biomass were better than that of N or P fertilizer being applied respectively. The optimal theoretical amount of N and P fertilizer being combination for the growth of E. maidenii seedlings was N fertilizer 0.60 g/plant and P fertilizer 0.66 g/plant. Applying N fertilizer and P fertilizer respectively had a significant promotion influence on the total N and P content of each organ (roots, stems, and leaves) of the seedling, however, applying combination of N fertilizer and P fertilizer had no significant promoting effect.