To reveal the biological characteristics of urban forest soil and the effects of soil enzyme on soil fertility as well as the correlation between physicochemical properties and enzyme activities, 44 urban forest soil profiles in Nanjing were investigated. Basic soil physicochemical properties and enzyme activities were analyzed in the laboratory. Hydrogen peroxidase, dehydrogenase, alkaline phosphatase, and cellulase were determined by potassium permanganate titration, TTC (CHN·Cl) colorimetry, phenyl phosphate dinatrium colorimetry, and anthrone colorimetry, respectively. The result showed that soil pH, organic carbon (C), and total nitrogen (N) had great effects on hydrogen peroxidase, dehydrogenase, and alkaline phosphatase activities in 0–20 cm thick soil. However, pH only had great effect on hydrogen peroxidase, dehydrogenase, and alkaline phosphatase activities in 20–40 cm thick soil. Hydrogen peroxidase, dehydrogenase, and alkaline phosphatase were important biological indicators for the fertility of urban forest soil. Both in 0–20 cm and 20–40 cm soil, soil enzyme system (hydrogen peroxidase, dehydrogenase, alkaline phosphatase, and cellulase) had a close relationship with a combination of physicochemical indicators (pH, organic C, total N, available K, available P, cation exchange capacity (CEC), and microbial biomass carbon (C)). The more soil enzyme activities there were, the higher the fertility of urban forest soil.