Abstract: In order to understand the effects of substrate, slow-release fertilizer (SRF), gibberllic acid (GA₃) and naphthalene acetic acid (IAA) on the biomass accumulation and water contents of Pinus yunnanensis seedlings, the U₁₅^*(5⁸) which deformed from the U₁₅^*(15⁸) uniform design was implemented the experiment. The findings indicated that mean biomass and water contents were 0.100 2−0.196 3 g/seedling and 69.8%−76.3% for 165-day-old seedlings, respectively, and there were significant differences of which the 2 parameters among the treatment combinations, the biomass and water content of the control presented significantly lower than or the smallest of which the other TCs. The SRF type was the dominant factor affecting the biomass and water contents, while the TC of the maximum biomass and the highest water content was fertilized the Aolü 5 (A5) granule SRF and SRF 5^#. The dominant factors affecting the biomass and water content of needles, stems and roots were not identical, with the SRF and GA₃ were the dominant factors affecting the biomass accumulation and increasing the water contents of the needles and roots, while the dominant factors influencing the which of the stem were the substrate and the SRF, respectively. The comprehensive optimal TC was the 0.15 g/L IAA solution pre-soaked the seed then sown it in the mixed substrate of 3 forest soils and 1 humus soil, and applied 1.0 g/container (2 seedlings/container) the SRF 5^#, in terms of this TC approach could raise sound seedlings in the practice. There was a linear relationship between the biomass and water content of whole seedling with the factorial levels Y=0.160+0.003x₁+0.007x₂−0.008x₃−0.004x₄ and Y=72.742+0.158x₁+0.135x₂+0.331x₃−0.705x₄, respectively. The biomass and water content of needles, stems and roots accounted for 69.2%, 19.4% and 11.4% and 74.6%, 19.4% and 6.0% which composited the whole seedling, respectively. The response of the biomass of P. yunnanensis seedling to root growth and development was more sensitive than that of growth parameters, and the accumulation of seedling biomass was selective to fertilizer types. The SRF with relatively long fertilizer effect was beneficial to the biomass accumulation and the water content increase of the organs.