In order to study the effects of bio-organic fertilizer on the fertility and enzymes activities in fluvo-aquic soil. A field experiment was conducted, and the changes of soil fertility characteristics and enzymes activities were measured in different growth stages of cucumber at 0—15 cm and 15—30 cm soil layers. The bio-organic fertilizer was once applied at rates of 0, 10 and 20 t/hm2, designated as CK, Y1 and Y2 respectively. The results showed that: (1) In the same growth period of cucumber, all the contents of total nitrogen, ammonium nitrogen, nitrate nitrogen, available phosphorus and organic matter increased at 0—15 cm and 15—30 cm soil layers with increasing bio-organic fertilizer application rates. In the mature period of cucumber, Y1 increased the contents total nitrogen, ammonium nitrogen, nitrate nitrogen, available phosphorus and organic matter in soil by 5.99%, 11.21%, 17.48%, 5.98% and 39.30% respectively, while the corresponding values were 6.59%, 25.86%, 21.99%, 10.78% and 61.76% respectively in Y2, compared with CK. Significant differences were found in ammonium nitrogen and organic matter among the three treatments. Under the treatments applied with bio-organic fertilizer at 15—30 cm soil layer, the nitrate nitrogen contents were increased by18.83% to 23.27%, and available phosphorus by 39.30% to 61.76%. (2) In the same growth period, the increased trends of soil urease and catalase activities were found with the increasing bio-organic fertilizer application rates at different soil layers. In the mature period, under the treatments applied with bio-organic fertilizer, soil urease activity was increased by 47.02% to 62.08%, and catalase activity by 14.41% to 23.73% compared with CK at 0—15 cm soil layer, while the values were 49.17% to 67.75% and 40.01% to 63.33% at 15—30 cm soil layer. (3) With the growing season, soil fertility indexes and urease activity decreased, while catalase activity increased first and then decreased. Besides, soil fertility indexes and enzyme activities at 0—15 cm soil layer were significantly higher than those at 15—30 cm soil layer. In conclusion, the soil fertility, urease and catalase activities were obviously increased in treatments applied with bio-organic fertilizer, and the increases in soil fertility, urease and catalase activities were found in fluvo-aquic soil with the increasing bio-organic fertility application rates.