OBJECTIVE： To study the interaction between bovine serum albumin （BSA） with lamivudine， efavirenz， tenofovir and its mechanism. METHODS： Fluorescence spectroscopy was used to determine the interaction between BSA with different concentrations of lamivudine， efavirenz， tenofovir under different temperatures. The fluorescence intensity of them were determined respectively； quenching constant （KSV）， apparent quenching constant （Kq）， binding constant （KA）， binding site （n）， thermodynamic enthalpy change （ΔH）， free energy diversification （ΔG） and entropy change （ΔS） were calculated according to Stern-Volmer equation and so on. Molecular docking model of 3 drugs with BSA was established by using Sybyl 6.7 Flex X model. RESULTS： Kq for the interaction between 3 drugs with BSA were all higher than 2.0×1010 L/（mol·s）， and were decreased with the increase of temperature； all n were close to 1， and thermodynamic functions ΔG＜0， ΔS＜0， ΔH＜0. Molecular docking model showed that 3 drugs were mainly bound with BSA at Sudlow Ⅰ subdomain site. CONCLUSIONS： There are the interaction between 3 drugs with BSA； fluorescence quenching mainly manifests as static quenching； binding reaction belongs to spontaneous molecular action process； binding force mainly includes hydrogen bond and Van der Waals’ force. The result of fluorescence experiment is consistent with those of molecular docking， and they complement each other.