The significance of sulphur (S) availability for crop yield and quality is highlighted under the global S deficiency scenario. However, little is known about the temporal trend in belowground organic S mineralisation when restoring land to productive agricultural systems, particularly for the deeper soil parts. Therefore, we investigated the decomposition of 35S-labelled methionine in surface (0-30 cm) and subsurface soil (30-60 cm and 60-90 cm) over a 48-year recultivation chronosequence (sampled after1, 8, 14, 24 and 48 years). Soil total sulphur (TS) significantly (p < 0.05) increased in surface soil but not in subsurface soils after 48 years of recultivation. Overall, the immobilisation of 35S-methionine (35S-MB) in subsurface soils relative to year 1 significantly decreased over the chronosequence but did not change in the surface samples. The 35S-MB values in subsurface soils were positively corrected with soil carbon (C) stoichiometry (Pearson correlation, p < 0.05), suggesting the immobilisation of methionine was likely constrained by microbial C demand in deep soil. Compared to year 1, 35S-SO42- released from 35S-methionine significantly declined throughout the older (≥ 8 years) soil profiles. Significant (p < 0.05) changes in the organic 35S partition (35S immobilisation and 35S released as sulphate) were observed in year 8 after the soil was recultivated with N-fixing alfalfa or fertilisers. Whereas, after that (≥ 14 years), soil organic S partition remained affected when conventional tillage and agricultural crops dominated this site. Indicating that the effect of recultivation on organic S decomposition depends on the manner of recultivation management. Our study contributes to an improved understanding of amino acid S and organic S mineralisation under severe anthropogenic disturbance.