Yak dung is an important factor affecting the nutrient cycling of soil in alpine grassland ecosystems, and changes in soil enzyme activity can effectively measure the soil nutrient cycling processes. To explore the temporal and spatial variations in soil enzyme activity under the addition of yak dung in alpine meadows, a fluorescence analysis method using 96-well microplate enzyme assays was employed. Key enzymes involved in soil carbon and nitrogen transformation processes in the alpine meadow soils of the eastern Tibetan Plateau：β-glucosidase (BG), peroxidase (PER), phenol oxidase (PPO), β-N-acetylglucosaminidase (NAG), and protease (LAP)—were studied to analyze the potential impacts of yak dung decomposition over different periods in warm and cold seasons. Also, the effect of the yak dung on the determined properties was considered at varying distances from the dung pile (under the dung (D0), 10 cm away (D10), and 20 cm away (D20)). The results indicate that (1) The decomposition of yak dung in both warm and cold seasons significantly increased the activities of BG, PER, PPO, NAG, and LAP, with the highest enzyme activity observed under D0. As the decomposition time progressed from warm to cold seasons and the distance from the dung increased, soil enzyme activity gradually decreased; (2) The decomposition of yak dung in both seasons significantly enhanced the total soil nutrients (total carbon, total nitrogen, total phosphorus) and available nutrients (ammonium nitrogen, nitrate nitrogen, available phosphorus), soil moisture, and pH, although the impacts of decomposition time on these soil environmental factors varied between seasons. The correlation between soil physicochemical properties and enzyme activity in the cold season was significantly stronger than in the warm season, with the C/N ratio in the cold season having the most pronounced effect on enzyme activity. The addition of exogenous nutrients led to the redistribution of nutrients and organic matter, with changes in enzyme activity exhibiting spatial and temporal gradient distribution characteristics, which were significantly correlated with the distance from the dung (radiating outward) and soil depth (extending downward).