Abstract: To reveal the anti-protein aggregation ability of brazilin (Bra), hematoxylin (Hto) and hematein (Hte) under physiological crowding environment, the macromolecular crowding reagent (polyethylene glycol) was used to construct a simulated crowding environment. Then the inhibition mechanisms of these three structurally similar polyphenol compounds on the aggregation behavior of human serum albumin (HSA) were investigated by fluorescence spectroscopy, UV-vis absorption spectroscopy, dynamic light scattering (DLS) and atomic force (AFM) microscope assay. The results showed that they could maintain the stability of the HSA structure, decrease the number of amyloid fibrils and shorten the length of aggregates in crowding environment. Therefore, they inhibited the aggregation process of HSA, and the sequence of the inhibition ability was in the order of Hto>Bra>Hte. Moreover, the presence of crowding reagents could lead to a decrease in the inhibitory activity of these three inhibitors compared to in vitro dilute solution. In conclusion, this study suggested that Hto could be used as a potential protein aggregation inhibitor and a functional food ingredient for the treatment and intervention of amyloid-related diseases.