The substrate enzyme activity and the spatial distribution of microorganisms are important factors affecting the degradation of pollutants in water treatment system of the constructed wetland. The present study using a laboratory scale constructed wetland system in three different conditions to measure the removal effects of nitrogen, the relationship between the removal efficiency and the spatial distributions of microorganisms and the substrate enzyme activity. Spartina alterniflora was selected as the plant of the constructed wetland, and the density of reed was 64 per square meter. The substrates were filled with fine sand, coral rock and blast furnace slag. In three different conditions, the removal rate of different forms of nitrogen, the quantity and activity of microorganisms, the substrate activity were investigated. The results showed that the average removal rate of TN and NH4-N were (25.02±12.69)% and (82.91±17.51)% in the constructed wetland system during two-month stable operation, respectively. The quantities of microorganisms in different depths were obviously different, and the activities of substrate urease and dehydrogenase were different with depths. The quantities of aerobic microorganisms, the activities of urease and dehydrogenase in the upper and middle layer were significantly higher than those in the lower layer of the system. In the down flow tank of the system, the quantities of nitrifying and nitrite bacteria in the upper and middle layer were higher than those in the lower layer; however, the distribution of denitrifying bacteria was opposite. There were positive correlations between the removal rate of nitrogen and the quantity of bacteria and the enzyme activity (R2=0.50 and 0.61, respectively). There was a significant positive correlation between the total nitrogen removal rate and the activity of urease (R2=0.86). The results contribute to the migration mechanism of nitrogen in the constructed wetland system of treating marine aquaculture waste waters.