Based on 24-year long-term field experiment established in 1990 in Yingtan Red Soil Ecological Experiment Station, the experiment included 4 treatments: CK(without fertilization), C(manure), NPK(N,P,K fertilizers), NPKC(NPK plus manure), soil samples were collected. The activities of invertase, urease (adding 0.5 ml toluene and not) and invertase kinetics were determined, and the soil nutrient contents and microbial biomass carbon were measured, in order to analyze the relationships among enzyme activity, soil nutrient and microbial biomass carbon to disclose the response of the activity of extracellular enzyme and nutrients to the long term fertilizations. Compared with CK, application of fertilizers increased the invertase activities by 31.3%–131.7% and microbial biomass carbon by 84.9%–125.1%. Without toluene inhibiting the microorganism activity, the invert rate of the substrate of invertase and urease significantly increased by 89.5%–153.7% and by 59.2%–98.9%, respectively; Microbial biomass carbon had significant positive correlations with the activities of invertase and urease, which imply microorganism significantly influenced the activities of these enzymes. Compared with CK, application of fertilizers increased significantly organic matter (by 30.1%–36.3%), total phosphorus (by 28.6%–102.9%), available phosphorus (by 62.2%–445.0%) and available nitrogen (by 35.9%–56.4%), the activities of soil urease and invertase were positively correlated with the contents of available nitrogen and organic matter. Compared with CK, application of fertilizers increased significantly the transfer coefficient (Vmax/Km) and maximum velocity (Vmax) but influenced little on the Michaelis constant (Km). Long term fertilization increased the soil nutrient contents and Microbial biormass carbon, which result in the increase of the activity of the extracellular enzymes.