The adsorption reaction of L-cysteine on Pd thin layer surface constructed by nano-dots under water environment has been investigated by S K-edge NEXAFS with He-path system under atmospheric pressure. It is revealed that L-cysteine physisorbs on Pd/Al2O3/NiAl(111) substrate surface through the thiol group and the carboxyl one, and chemisorbed L-cysteine exists on the surface as L-cysteine thiolate. Before and after dipping the Pd/Al2O3/NiAl(111) substrate into L-cysteine aqueous solution, the surface morphology is observed using AFM. The morphology of the Pd/Al2O3/NiAl(111) substrate is composed of many nano-dots before dipping. After dipping, the nano-dots are enlarged.