Taking the subtropical pine plantation as research object, the soil biochemical properties and enzymes activities were studied using three simulated sulfur deposition experiments with different levels (control treatment, CK:pH 6.5; low sulfur treatment, LS:pH 4.5; high sulfur treatment, HS:pH 2.5). The results showed that:(1) Sulfur input had a significant effect on soil acid, HS significantly reduced the 0-5 cm soil pH, both LS and HS treatments could significantly reduced the 5-10 cm soil pH (P<0.05). (2) Sulfur input had a certain effect on soil organic carbon pool. Soil total organic carbon (TOC) had no significant response to sulfur input, but its differences increased in soil layers (P<0.05). The impact of sulfur input on soil dissolved organic carbon (DOC) was limited. HS significantly decreased soil microbial biomass carbon (MBC) from 5-10 cm soil layer and increased the differences in soil layers (P<0.05). (3) Sulfur input had different effect on soil effective nitrogen pool. Soil dissolved organic nitrogen (DON) and ammonium nitrogen (NH₄⁺-N) had no significant change. HS treatments significantly decreased soil nitrate nitrogen (NO₃^--N) and soil microbial biomass nitrogen (MBN) and increased the differences in soil layers (P<0.05). (4) Sulfur input inhibited the activity of soil enzyme. HS significantly decreased soil urease activity (P<0.05). Soil invertase activity had no significant changes, but sulfur input reduced the differences of enzymes activities in the two soil layers. The comprehensive analysis of soil biochemical characteristic and enzymes activities indicators from all treatments showed that soil pH and enzymes had sensitive responses to sulfur input. Soil layer was another major impact factor. But the interaction between sulfur addition and soil layer had the limited impact. Through the Pearson method, sulfur input changed the correlation of the soil biochemical properties and enzymes activities. The effect of simulated acid rain on soil acidification was a gradual process of accumulation. The effect of exogenous sulfur addition on soil carbon, nitrogen and enzymes activities was different. The results could provide scientific basis for forest management under environmental stress of sulfur deposition.