N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-1-(2-fluorophenyl)-4-methyl -5-oxo-4,5-dihydro-1H-1,2,4-triazole-3-carboxamide(LJC-116) is a kind of new Type Ⅱ compounds,c-Met kinase inhibitors with 4-phenoxy quinolone.In vitro experiments showed that they have an excellent anti-tumor activity.In this paper,the in vitro molecular binding interaction between LJC-116 and human serum albumin(HSA) was further investigated by various methods,such as fluorescence spectrometry,synchronous fluorescence spectrometry,fluorescence probe experiments,three-dimensional(3-D) fluorescence spectrometry,ultraviolet-visible(UV-Vis) absorption spectroscopy and molecular docking.It was found that the intrinsic fluorescence of HSA could be quenched by LJC-116 through a static quenching process.The static quenching process was also proved by 3-D fluorescence spectrometry and ultraviolet-visible absorption spectroscopy.The apparent binding constants between LJC-116 and HSA at 288,299，310 K were estimated to be in the order of 104 L·mol-1,which indicated the binding between LJC-116 and HSA was moderate.The thermodynamic parameters ΔH°,ΔG° and ΔS° were calculated,in which the negative ΔG° suggested that the binding of LJC-116 to HSA was spontaneous.Moreover,the positive ΔS° and negative ΔH° revealed that hydrophobic interaction and hydrogen bonds were the major forces to stabilize the protein- LJC-116(1∶1) complex.Under the experimental conditions,the overlapping integral J=7.169 7×10-15 cm3·L·mol-1，R=1.28 nm，r=1.69 nm,E=15.6% were obtained.It indicated a high probability of energy transfer from HSA to LJC-116.Probe agent ibuprofen and warfarin were added in system of HSA-LJC-116,it showed that the primary binding site of LJC-116 was located at site Ⅰ(subdomain ⅡA) of HSA.The slight redshift of fluorescence(0.3 nm),synchronous fluorescence(0.4 nm) and 3-D fluorescence spectra(2 nm) all demonstrated that the polarity of microenvironment for amino acid residues in HSA increased and the hydrophobic property decreased.Finally,the forces deduced from the thermodynamic calculation,and the conformational changes of HSA were validated by the molecular docking.The interaction mechanism of HSA and LJC-116 was comprehensively expatiated at a molecular level in this paper,and some useful information for the transport of Type Ⅱ c-met kinase inhibitors in vivo was provided.