||Purpose Soil enzymes are the main drivers of microbial catabolic and anabolic processes. However, the response of functional enzymes strategies and microbial metabolic efficiency (MME) to nutrient stoichiometry remains unclear in straw-amended black soil. Methods Four nutrients gradients of N0, N1, N2, and N3 were set, and N0-N3 mean straw-amended soil with increasing addition of nitrogen (N), phosphorus (P), and sulfur (S). The activities of five enzymes involved in carbon (C), N, and P acquisition were investigated under different nutrient treatments in two soils during an 84-day incubation study. Microbial nutrient availability and C use efficiency (CUE) were assessed based on enzymatic stoichiometry (ES), and their potential relationship to change net soil-C (Net-C) was then established. Results Compared to soil with straw alone, the Net-C in Gongzhuling and Hailun increased by 1.2-2.3 and 1.3-1.9 times with stoichiometric nutrient management, respectively. ES revealed that microbial metabolism was successively limited by N and P in Gongzhuling soil and was mainly limited by P in Hailun soil. Nutrient supplementation improved the MME, supported by a decrease of - 9.2% (- 3.4 to - 17.8%) in qCO(2) and an increase of 10.1% (2.9-23.6%) in CUE. Path analysis suggested that nutrient supplementation influenced Net-C by directly and positively regulating C and P acquisition EEAs and CUE. The Net-C in Gongzhuling was more sensitive to nutrient supplementation, while that in Hailun was more limited by P, indicating that microbial metabolism might be related to soil pH or clay content. Conclusions Combined addition of straw and N, P, and S improved Net-C by alleviating microbial nutrient limitation and improving CUE, in which process P acquisition enzyme, P availability, and soil properties (pH or clay content) played a key role.