The interaction between chitosan (CS), its complexes CS-La and CS-Dy, and bovine serum albumin (BSA) in vitro under simulative physiological conditions was studied by fluorescence, ultraviolet-visible absorption and synchronous fluorescence spectrometry. The results showed that CS, CS-La, CS-Dy had strong effects on quenching of the fluorescence launching and enhanced the UV absorption spectra of BSA. After the fluorescence quenching, the obtained data were analyzed by Stern-Volmer equation, the results indicated that the reactions between BSA and CS, CS-La and CS-Dy generated new complexes. The quenching belonged to static fluorescence quenching, with non-radiation energy transfer happening within single molecule, but the static and dynamic quenching coexisted in the reactions between CS and CS-Dy and BSA. The binding constant KA (291 K, CS-BSA: 1.807′104 [L/(g·s)], CS-La-BSA: 3.065′104 [L/(g·s)], CS-Dy-BSA: 2.193′104 [L/(g·s)]; 310 K, CS-BSA: 2.665′104 [L/(g·s)], CS-La-BSA: 2.022′104 [L/(g·s)], CS-Dy-BSA: 7.246′104 [L/(g·s)] and thermodynamics parameters (DH, DS, DG) were calculated, respectively according to the equation of fluorescence spectrometry at different temperatures. Based on thermodynamic data, the main reactions between CS and CS-Dy and BSA were electrostatic and hydrophobic forces, but those between CS-La and BSA van der Waals and hydrogen bond forces. The effects of CS, CS-La and CS-Dy on the conformation of BSA shown by synchronous fluorescence spectrometry indicated that they changed the micro-environment and conformation of BSA molecules.