Extracellular enzymes are the major mediators of plant residue and organic matter decomposition in soil, frequently associated with microbial metabolic processes and the biochemical cycling of nutrients in soil ecosystems. However, the dynamic trends and driving factors of extracellular enzymes and their stoichiometry during plant residue transformation remain to be further studied. Here, we investigated the dynamics of extracellular enzymes and enzymatic stoichiometry in the “litter-soil” transformation interface soil (TIS) layer, an essential occurrence layer for microbially-mediated C transformation. The results indicated an unbalanced relationship between substrate resource supply and microbial metabolic demand. Microbial metabolism was limited by C (C/N-acquiring enzymes > 1) and P (N/P-acquiring enzymes < 1) throughout the observed stages of plant residue transformation. The initially higher extracellular enzyme activity reflected the availability of the active components (dissolved carbon (DC), nitrogen (DN), microbial biomass carbon (MBC), nitrogen (MBN), and phosphorus (MBP)) in the substrate and the higher intensity of microbial metabolism. With the transformation of plant residues, the active fraction ceased to be the predominant microbial C source, forcing the secretion of C-acquiring enzymes and N-acquiring enzymes to obtain C sources and N nutrients from refractory substrates. Moreover, C/N-acquiring enzymes decreased, while C/P-acquiring enzymes and N/P-acquiring enzymes subsequently increased, which suggested that the microbial demand for N gradually increased and for P relatively decreased. Soil microorganisms can be forced into dormancy or intracellular mineralization due to the lack of substrate resources, so microbial biomass and extracellular enzyme activities decreased significantly compared to initial values. In summary, the results indicated that soil nutrients indirectly contribute to extracellular enzymes and their stoichiometry by affecting microbial activities. Furthermore, extracellular enzymes and their stoichiometry were more sensitive to the response of soil microbial biomass carbon.