Abstract: The effects of addition of biochar on the matrix enzymes activity, microorganisms population and microbial community structure were evaluated under cucumber continuous cropping for 6 years (11 rotations). Cucumbers were grown in pots in greenhouse with 5% or 3% of medium (by mass) substituted with biochar. The control consisted of medium alone without biochar. The results showed that the activity of peroxidase was significantly improved to the level of the first rotation crop form 30 to 120 d after planting in both biochar treatments, with the effect of 5% biochar being more significant than that of 3% biochar. However, the neutral phosphatase activity was markedly reduced after biochar treatment. The addition of 5% biochar had significant regulation effect on the activities of invertase and urease from 30 to 90 d after planting, while the addition of 3% biochar had little effect. The populations of bacteria and actinomycetes were increased and the fungi population was reduced in both biochar treatments from 30 to 90 d after planting, and the effect of 5% biochar was more significant than that of 3% biochar. Meanwhile, the addition of biochar significantly increased the diversity of the bacterial community structure. In summary, biochar had obvious regulation effect on soil enzyme activity, microorganism quantity and microbial community in continuous cropping nutrition medium.