Water and fertilizer are the two main factors promoting the fast growth and high-yielding of Populus tomentosa, and thus, to study their coupling effects on the biomass of P. tomentosa seedlings has important practical significance. Taking the P. tomentosa clone 87 seedlings as test materials, a pot experiment with rotary combination design of square regression of three factors with five levels was conducted in the nursery of Beijing Forestry University from March to October 2008 to study the coupling effects of water, fertilizer N, and fertilizer P on the biomass of the seedlings, and a related regressive mathematical model was established. The results showed that water was the main factor affecting the biomass of P. tomentosa seedlings, followed by fertilizer N, and fertilizer P. With the increasing input of the three factors, the biomass of the seedlings increased, but when the input was beyond a certain level, the biomass began to decrease. There was a significant positive interactive effect between water and fertilizer N, but a less interactive effect between fertilizer N and fertilizer P and between water and fertilizer P. In our case, the optimal combination of water and fertilizer was 73.37% of field capacity + 4.14 g x plant(-1) of N fertilization + 1.41 g x plant(-1) of P fertilization, under which, the biomass of P. tomentosa seedlings achieved 68.30 g x plant(-1).