The effects of different amounts of biochar on the growth of Eucalyptus spp. seedlings and soil enzyme activities were investigated，in order to obtain the optimal substrate for Eucalyptus spp.seedling nursery，so as to provide theoretical reference for the rational application of biochar.The experiment was conducted with Eucalyptus spp.seedlings in container nursery substrates with different mass ratios of biochar，and six gradients of 0(CK)，2%(T1)，4%(T2)，6%(T3)，8%(T4)and 10%(T5)were set up，with three replications for each treatment，and a completely randomized design was adopted.The growth of Eucalyptus spp. seedlings，soil chemical properties and soil enzyme activities in the various treatments were analyzed.The results showed that:(1)4%-6% biochar increased the growth of Eucalyptus spp. seedlings.(2)Biochar application improved soil nutrients of Eucalyptus spp. substrate in different degrees，and the trends of pH，soil organic matter，total nitrogen，and available potassium with the increase in the amount of biochar application were consistent as follows:T5>T4>T3>T2>T1>CK，and the available nitrogen，available phosphorus，and cation exchangeable capacity contents were significantly increased by 9.25%，119.51%，and 108.77%，respectively，compared with CK，while other indexes did not differ significantly from CK.(3)The activities of soil catalase，β-glucosidase，acid phosphatase，and sucrase were reduced with the increase of biochar application.The activity of soil urease increased with the increase of biochar application，and the highest content was found at 10% of biochar application.(4)The nutrient indexes of Eucalyptus spp. seedlings with different additive treatments had four principal components with eigenvalues greater than 1，and the cumulative contribution rate reached 82.754%，the main influence factor of the first principal component was catalase，and the main influence factors of the second principal component were total phosphorus and soil organic matter；the third principal component main influence factor was available nitrogen；and the fourth principal component main influence factor was total potassium.In general，application of 4%-6% biochar could significantly improve the growth of Eucalyptus spp. seedlings，and increase the content of nutrient elements in seedling substrate and the activities of main soil enzymes.