One-year-old seedlings of Fengyang No.1were chosen as experimental materials, which were grown in six substrates with different water contents by weighting method (CK, T1, T2, T3, T4 and T5), and the difference of physiological and biochemical characteristics of C. oleifera seedlings were determined and analyzed. The results were as follows. (1) The leaf water content and the ratio of free water to bound water in CK group were the largest, and the values were 68.5% and 26.4, respectively. However, those in the other four treatments declined with the decrease of substrate water content. (2) The relative conductivity, POD activity and MDA content decreased first, and then increased with the decline of substrate water content. In addition, those values in T1 group were the smallest, being 37%, 11.1 U·min^-1·g^-1 and 0.3 μg·g^-1, respectively. (3) The content of free proline content and soluble sugar rose with the decline of substrate water, which varied from 231.4 μg·g^-1 and 681.4 μm·g^-1 in CK group to 40.4 mg·g^-1 and 48.9 mg·g^-1in T4 group, respectively. (4) When the substrate water content was above 90%, the reducing of leaf stomatic aperture was not beneficial for photosynthesis and respiration of the seedling. While the substrate water content dropped to less than 81%, the seedlings would suffer drought stress. Therefore, substrate water content of 81%-90% was the most suitable for seedlings growth. The substrate water content should be controlled in the appropriate range to keep seedlings growing normally in C. oleifera production.