In this paper, black soil samples at the depths of 0-20 cm and 20-40 cm were collected from the Hailun Agricultural Ecology Station of Chinese Academy of Sciences to study the effects of long-term fertilization on their urease, invertase, phosphatase and catalase activities and total C and N contents. The results showed that long-term application of chemical fertilizers and organic manure increased the activities of urease, invertase and phosphatase in 0-20 cm and 20-40 cm soil layers in different degree, and the combined application of them increased the activities of the three enzymes significantly, with an increment of 43.6%-113.2%, 25.9%-79.5% and 14.7%-134.4% in 0-20 cm soil layer and 56.1%-127.2%, 14.5%-113.8% and 16.2%-207.2% in 20-40 cm soil layer, respectively. However, long-term application of chemical fertilizers without organic manure had little effects on catalase activity. The activities of urease, invertase and phosphatase decreased with increasing soil depth. Long-term application of N fertilizer increased urease activity, and P fertilization had obvious positive effect on phosphatase activity. Long-term fertilization also had obvious effects on the soil total C and N contents and C/N ratio.