Leucomalachite Green (LMG) is a major metabolite of malachite green (MG). It has a long residence period in edible fish tissues. At present, the use of MG has been banned in some countries for its increased risk of carcinogenesis, mutagenesis and other adverse effects to human health. However, MG is still widely used in aquaculture, aquatic transport and storage for its low price. The interaction between LMG and bovine serum albumin (BSA) under simulative physiological conditions was investigated by spectroscopy. Two spectroscopic approaches (fluorescence and circular dichroism) and two different experiments were used for monitoring the biological dynamic process. Qualitative and quantitative information was obtained with the resolution of the data matrices by chemometrics method - multivariate curve resolution-alternating least squares (MCR-ALS). Atomic force microscope (AFM) was executed in order to verdict the particle morphology and dimensions of the LMG-BSA conjugates. The root mean square (RMS) roughness of the individual BSA molecule was found to be (1.24±0.28) nm. The BSA molecule particle was observed to be looser on the mica substrate upon interaction with LMG. The RMS was changed to be (13.47±0.53) nm for the LMG-BSA interaction. The calculated result of circular dichroism (CD) spectra revealed that the α-helical content for the LMG-BSA complex was 42.5%, which has a slight decrease compared with the free BSA (46.3%). The results of AFM and CD spectra showed that the binding of LMG to BSA induced micro-environmental and conformational changes of BSA molecules. In order to identify the LMG-binding site on BSA, site marker competitive experiments were carried out, using drugs which were specifically bound to site Ⅰ (warfarin) and site Ⅱ (ibuprofen) on BSA. The binding constant of the system with warfarin (1.88×106 L·mol-1) was almost 70% of that without warfarin (2.65×106 L·mol-1), while the constants of the systems with and without ibuprofen had only a small difference, indicating that LMG was bound to site Ⅰ of BSA. The molecular docking gave more intuitive understanding of the binding of LMG and BSA. It was recognized that LMG binds within the sub-domain ⅡA pocket in domain Ⅱ of BSA. These values showed that hydrophobic forces were the main interactions in the binding of LMG to BSA and the stabilization of the complex. It can be expected that the study will have great significance in helping to further clarify the metabolism and distribution of LMG in vivo and the mechanism of toxicological effects and pharmacokinetics from molecular level.