The main objective of this study was to study the effect of L-cysteine and nanoemulsification on the bioaccessibility, release, solubility and digestion of β-sitosterol in the simulated gastric- and intestinal fluids (SGF and SIF). In general, nanoemulsification of β-sitosterol in the double layer led to enhance its solubility and digestion in SGF and SIF. The current study showed that the bioaccessibility of β-sitosterol W/O/W nanoemulsion showed a considerable increment from 44.23% to 54.18% by increasing L-cysteine content from 0.25% to 1%. In this regard, the average droplet size increased from 0.149 to 0.152 �m in SGF. The bioaccessibility of β-sitosterol was improved up to 78.1% after 2 h incubation during the second stage of digestion in SIF. The solubility of β-sitosterol nanoemulsion in SIF was increased from 70.03% to 92.20% by increasing L-cysteine concentration from 0% to 1%. The droplet flocculation occurred in SGF (after 60 min digestion). The current study confirms the synergistic role of L-cysteine in improving the solubility and bioaccessibility of β-sitosterol in the SGF and SIF