In this study, the binding behavior of β‐sitosterol with lysozyme (LZM) was elucidated by surface plasmon resonance (SPR), computational molecular docking and molecular dynamics simulation studies. Chicken egg white lysozyme (CEWLZM) served as a model protein. Tri‐N‐acetylchitotriose (NAG3) was used in the redocking experiments to generate precise binding location of the protein. β‐sitosterol displayed a slightly better binding energy (‐6.68±0.04 kcal/mol) compared to NAG3. Further molecular dynamics simulations and MMPBSA analysis revealed that residues Glu35, Gln57‐Asn59, Trp62, Ile98, Ala107 and Trp108 contribute to the binding energy. Then, 2.5 mg/mL CEWLZM, 1X PBS buffer (pH 7.4) as running and coupling buffers, 30 µL/min as flow rate were applied for SPR analysis. Serial β‐sitosterol injections (20‐150 μM) were performed through SPR sensor surface. According to SPR binding study, KD value for β‐sitosterol‐CEWLZM binding interaction was calculated as 71.34±9.79 µM. The results could provide essential knowledge for nutrition, pharmaceutical science, and oral biology.