Soils are the main component of the urban ecosystem and they perform environment-forming and protective functions. The dynamics of the organic matter and enzymatic activity is an early diagnostic indicator of negative changes in urban pedogenesis. However, the features of the enzymatic activity of urban soils of the Chernozem zone are poorly studied, and the available research focuses on the upper humus-accumulative horizons or the urbic horizons. The study aimed to reveal the regularities of the enzymatic activity distribution through 200 cm in the profile of urban soils and to show their relationship with the content of the main soil components. Urbic Technosols Molic, Ekranic Technosols, Urbic Technosols, Technic Chernozems (caChernozems of fallow and virgin areas of the Rostov Agglomeration were studied. Soil samples were taken down to a depth of 200 cm. The activity of extracellular enzymes was determined by the fluorescence method. Soil organic matter content was determined by the high-temperature combustion method on a TOC analyzer. А number of patterns in the distribution of the enzymatic activity have been identified. Most of the Urbic Technosols were characterized by a low enzymatic activity. Factor analysis showed a negative correlation of oxidative and hydrolytic enzymes in natural soil. A positive correlation of oxidases with hydrolases was shown in urbanized horizons. We found that the longer the time of sealing, the stronger the inhibition of enzymatic activity. We conclude that enzymatic activity can be used as an indicator in biodiagnostics, biomonitoring, and bioindication of the urban soils. © 2020 John Wiley & Sons, Ltd.