This study presents the results of a 13 years-monitoring of the soil enzyme β-glucosidase (C cycle) and the microbial biomass carbon (MBC) in three adjacent areas under no-tillage (NT), conventional tillage (CT) and under native cerrado vegetation.  The soil was classified as a clayey Typic Haplustox and the CT and NT areas were in a corn/soybean rotation. The experiment began in 1992 and it’s monitoring in 1999. In phase 1 (1999-2003) samples were collected at 0 to 5cm depth and in phase 2 (from 2004-2012) at 0 to 10cm depth. On average, in phase 1, β-glucosidase activity under NT was 2.8 times higher than at the CT and native cerrado. In phase 2, the magnitude of these differences fell to 1.7 times. For the MBC the difference between NT and CT was 1.9 times in phase 1, while it was 1.4 times in the second phase. Regarding the native cerrado vegetation, there were reductions in MBC in the two cultivated areas. Over the 13 years of monitoring in the three areas the differences obtained with the enzyme β-glucosidase were more pronounced. The differences between the areas were higher in phase 1 (0-5cm) than in phase 2 (0-10cm) and this effect is related to the stratification of the microbiological properties on the soil profile when there is no inversion of layers (NT). Therefore, the adoption of NT causes significant changes in the biological functioning of the soil, with a higher concentration of microbial activity in the surface layers. These results demonstrate the positive effect of the absence of soil disturbance under NT on the accumulation of MBC and β-glucosidase activity. Due to its sensitivity, ease of measurement and low cost, β-glucosidase activity holds potential for its use as a biological indicator of soil quality.