Temperature, moisture, and other environmental factors change with altitude, which directly or indirectly can affect soil microbial biomass, community structure, and soil enzyme activity. However, research on the response of soil enzyme activities in the alpine forest ecosystems of the subtropical regions is relatively limited. Daiyun Mountain is the largest Pinus taiwanensis germplasm resource in China. In the present study, Pinus taiwanensis at altitudes of 1300 m(L), 1450 m(M), and 1600 m(H) in the mid-subtropical Daiyun Mountain was used to study changes in soil microbial biomass and soil enzyme activities at different altitudes. The results showed that the effect of different altitudes on enzyme activity was generally weak in the B horizon, and cellulolytic enzymes activities (e.g., β-glucosidase (βG) and cellulolytic enzyme (CBH)) decreased significantly with increasing altitude in the A horizon, thus resulting in a decrease in soil soluble carbon (DOC) and microbial biomass carbon (MBC) with increasing altitudes. Although acid phosphatase activity (ACP) increased significantly with altitudes, there was no significant change in available phosphorus (AP) levels. In addition, microbial biomass nitrogen (MBN) and microbial biomass phosphorus (MBP) decreased significantly with increasing altitude. Redundancy analysis (RDA) showed that MBP and carbon/nitrogen (C/N) ratio were the most important factors determining soil enzyme activity in the A horizon, whereas soil water content (WC) and MBP levels played a major role in determining soil enzyme activity in the B horizon. This study showed that. in the phosphorus-limited subtropical region, inorganic phosphorus is easily fixed by iron and aluminum. MBP, can supplement available phosphorus in soil and become a major factor affecting enzyme activity in this area. Soil organic carbon and nitrogen decomposition related enzyme activities increased as decreasing altitude, which accelerated the turnover of soil carbon and nitrogen. Therefore, exploration of changes in enzyme activity at different altitudes provides a scientific basis for predicting soil carbon, nitrogen, and phosphorus cycling in mid-subtropical subalpine forest ecosystems, which provides a scientific basis for the management of Pinus taiwanensis forests in Daiyunshan Nature Reserve.