Aim To investigate the binding mechanism of fatty acid translocase FAT/CD36 and long chain fatty acid. Methods ELISA was used to analyze the intensity mechanism of the purity of FAT/CD36 binding with different fatty acid, molecular simulation docking was used to further verify the binding mechanism and analyze its possible binding sites. Results ELISA proved that the recombinant protein FAT/CD36 with different types of long chain fatty acids showed specific binding, binding values for oleic acid＞palmitic acid＞palmitoleic acid＞myristic acid＞stearic acid. Molecular simulation docking further confirmed that FAT/CD36 receptor protein mainly combines with oleic acid, palmitic acid and palmitoleic acid. And the binding site was located in the extracellular structure of the FAT/CD36 receptor protein Arg183-Lys-Gly-Lys-Arg-Asn-Leu-Ser-Tyr238 region consisting of the active pocket. Conclusion FAT/CD36, as the main receptor of long chain fatty acid transport, may transport oleic acid, palmitic acid and palmitoleic acid.