Cysteine (Cys) and methionine (Met) are unique amino acids that contain not only nitrogen (N) but also sulfur (S); thus, they are a source of S for plants under low-soil-SO42- conditions. However, whether low-molecular-weight organic N and S can be utilised by plants or contribute to plant growth remains unknown. Therefore, this study aimed to examine the uptake of organic and inorganic N and S by plants and the role of microbial decomposition under monocropping and intercropping based on 13C, 15N, 14C, and 35S quad labelling. As indicated by the 13C/14C uptake, clover, millet, and ryegrass utilised 0.24–1.68% of the added Cys and Met within 6 h and encountered considerable competition from soil microorganisms. The added Met and Cys were rapidly utilised by microorganisms, and part of the N was subsequently released as inorganic N, which was taken up by plants (15N-Cys: 9.3–15.2%; 15N-Met: 5.9–13.4%) within 6 h. Six hours after addition, 57.8–78.5% of the 35S-Met and 26.2–56.0% of the 35S-Cys were retained in the microbial biomass, while more 35S-Cys was mineralised to SO42−. Plants took up 5.5–12.4% of 35S-Cys and only 3.4–6.0% of 35S-Met, and 35S uptake was dominated by inorganic S after the mineralisation of Cys and Met. N uptake from Cys and Met accounted for less than 1% of the total N uptake from the soil, while S uptake from Cys and Met accounted for 9.3–27.0% and 2.8–11.8% of the total S uptake from the soil, respectively. Additionally, Cys was more rapidly mineralised to SO42− by soil microbes than Met; the produced SO42− was further utilised by plant roots. The contributions of Cys and Met to the total N and S uptake were the highest in millet monocropping while intercropping altered the relative contributions of organic and inorganic N and S. Overall, soil soluble Cys and Met played a limited role in plant N uptake but were an important source of plant S uptake.