Anthropogenic nitrogen (N) enrichment is a concern worldwide, as it affects almost every aspect of ecosystem function and composition. Many nitrogen (N) addition experiments have been well documented to decrease plant biodiversity across various terrestrial ecosystems. However, such generalizations about the impacts of nitrogen addition on soil enzyme activities and microbial biomass are lacking, especially in the Three Gorges Reservoir area, as a key sensitive ecological barrier of the middle and lower reaches of the Yangtze River. Therefore, we conducted nitrogen addition experiments in February 2019 in Pinus massoniana plantation in the Three Gorges Reservoir area of subtropical China. Soil samples were collected from three control plots (N_CK), three low-nitrogen addition plots (N_low), three middle-nitrogen addition plots (N_middle), three high-nitrogen addition plots (N_high) in May, August and November, 2019. We studied the soil enzyme activities, microbial biomass and nutrient content in response to nitrogen addition, which provided a theoretical basis for predicting soil seasonal dynamics in an increasing atmospheric nitrogen deposition background in the Three Gorges Reservoir area. The results show that, in the early stage of nitrogen addition, the medium nitrogen (60 kg hm^-2 a^-1) treatment increased the activities of β-1-4 glucosidase (BG), N-acetylglucosaminosidase (NAG), acid phosphatase (AP), polyphenol oxidase (PPO) and peroxidase (POD); the high nitrogen (90 kg hm^-2 a^-1) treatment increased the contents of soil organic carbon, total nitrogen, microbial biomass carbon (MBC), nitrogen (MBN), phosphorus (MBP), and the activities of AP, PPO, while decreased the soil pH, total phosphorus content and the activities of BG and NAG. There were significantly seasonal differences between soil enzyme activities and microbial biomass. Hydrolase activities, MBC and MBP contents in autumn were significantly higher than those in spring and summer, while oxidase activities and MBN contents were higher in spring and summer. Soil enzyme activities were significantly correlated with season, soil moisture content, soil nutrient, MBC, MBN, MBP. The change of soil enzyme activities is due to the comprehensive action of multiple factors. Redundancy analysis (RDA) showed that soil moisture content, MBC, MBN, MBP and total N greatly accounted for the variation in soil enzyme activities. The increasing nitrogen deposition will accelerate the formation of soil humus, increase organic carbon accumulation, lead to soil acidification, and produce phosphorus limitation in the local Pinus massoniana plantation.