Purpose <p>Sulfur deficiency in plants has become a global concern as a result of reduced sulfur input from non-sulfur fertilizers and decreased atmospheric sulfur deposition. However, few studies have simultaneously analyzed the responses of both plants and microorganisms to sulfur application. This study aimed to investigate the impact of sulfur application on paddy rice, microbial sulfur-acquiring enzyme synthesis, and microbial biomass sulfur.</p> Methods <p>To evaluate the effects of sulfur fertilization on both soil microorganisms and paddy rice, a pot experiment was conducted under field conditions using two soil types, with and without sulfur application.</p> Results <p>Sulfur application increased the sulfur concentration of straw by an average of 18.4% across all soils and enhanced the dry weight of straw and grain by 15.2% and 6.9%, respectively, in Fluvisols. It also more than doubled microbial biomass sulfur while decreasing the ratios of microbial biomass carbon to sulfur and arylsulfatase to β-D-glucosidase activities by 69.4% and 16.9%, respectively. Microbial biomass carbon/sulfur ratio negatively correlated with straw sulfur concentration and sulfur uptake in paddy rice, suggesting that this ratio reflects the sulfur status of paddy rice. Additionally, the effect size of the arylsulfatase/β-D-glucosidase ratio to sulfur application exhibited significant negative correlations with those of straw sulfur concentration and sulfur uptake in paddy rice, indicating that microbial arylsulfatase synthesis could reflect the sulfur response of paddy rice.</p> Conclusions <p>Therefore, the carbon-to-sulfur stoichiometry of microbial biomass and ecoenzyme activity may serve as a complementary indicator for evaluating sulfur status of paddy rice.</p>