A fluorogenic model substrate, methylumbelliferyl-N-acetylglucosamine was used to measure N-acetylglucosaminidase activities in wetland soils. The enzyme activity exhibited substrate saturation around 200 μM, and an approximate linearity for at least 90 min incubation time. The effects of temperature, pH, toluene, and γ-radiation on the enzyme were also determined. The method was applied to 32 different wetland samples from both freshwater wetlands and salt-water wetlands. Overall, the activities were the lowest in bogs and much higher in freshwater marshes and flooded grasslands. The variations of the activity were not explained by a single environmental variable. However, among peatland samples (e.g., bogs and fens) only, specific enzyme activity per organic matter exhibited a significant correlation with N-acetylglucosaminidase activity, suggesting the importance of the enzyme activity in organic matter dynamics in such systems.