Abstract: In order to study the interaction between Latifoloside G, Latifoloside C and Kudinoside G and bovine serum albumin (BSA) at different temperatures, and to provide experimental basis for the study of the transport and mechanism of Latifoloside in vivo, this paper used fluorescence spectroscopy to study the binding mechanism, binding mode, binding constants and binding sites of small molecules and proteins, and used circular dichroism to study the conformational changes of proteins. The results showed that all three saponins can effectively quench the endogenous fluorescence of BSA. Kudinoside G was static quenching, andLatifoloside G and Latifoloside C were dynamic quenching. With the increased of the concentration of the three small molecules, the endogenous fluorescence intensity of BSA decreases and the maximum emission peak of BSA was slightly blue-shifted (from 347 nm to 345 nm) at both temperatures, and the order of the binding ability of three saponins to BSA could be determined as Latifoloside G>Latifoloside C>Kudinoside G. The main types of forces between Kudinoside G and BSA were hydrogen bonding and Van der Waals forces, and the main forces between Latifoloside G and Latifoloside C and BSA were hydrophobic. The binding capacity of Latifoloside G and Kudinoside G with BSA was related to the polar group connected to C-28, and then Latifoloside C of alkane type was easier to insert into the hydrophobic cavity of BSA than Kudinoside G. The circular dichroism spectrum showed that the combination of three saponins with BSA could change the internal structural environment of BSA, the α-helix content increases, the microenvironment polarity decreases, and the hydrophobicity increased.