Soil enzymes act as crucial roles not only in participating in carbon and nutrient cycles, but also in indicating soil productivity and environment change. As a result, understanding the changes of soil enzyme activity can provide with key scientific basis for managing soil. Yet, little attention has been given to the differential responses of enzyme activity in soil organic layer (OL) and mineral soil layer (ML) to the seasonal fluctuations of environmental factors. An in-situ soil core incubation experiment was therefore conducted in the Masson pine (Pinus massoniana) plantation in the hilly area at the upper reaches of the Yangtze River. The activities of soil invertase, urease and acid phosphatase in both OL and ML were measured at early rainy season (ERS), mid rainy season (MRS), late rainy season (LRS), early dry season (EDS), and late dry season (LDS) from June, 2014 to April, 2017. The soil enzyme activities in both two layers changed significantly with the critical periods, and on the whole, the activities of the measured enzymes in the rainy season were higher than those in the dry season. The activities of invertase and urease in the OL decreased significantly with incubation year, while soil acid phosphatase activity in mineral soil layer increased with incubation year. The activities of invertase and acid phosphatase in the OL were significantly higher than those in the ML. The fluctuation ranges of all soil enzyme activities in the OL were higher than those in the ML. Partial least squares (PLS) analysis showed that soil moisture, microbial biomass, and substrate content had significant effects on soil enzyme activities, and their size of effects were strongly depended on the soil layer and enzyme type. Briefly, the responses of enzyme activities in the OL to periodically environmental fluctuation are more sensitive than those in the ML, and the dominant factors driving the dynamics of enzyme activities in the OL differ from those in the ML, implying that well managed soil organic layer in the plantation is a key to maintain and improve soil productivity.