Molecular docking is a widely used method to predict the binding modes of small-molecule ligands to the target binding site. However, it remains a challenge to identify the correct binding conformation and the corresponding binding affinity for a series of structurally similar ligands, especially those with weak binding. An understanding of the various relative attributes of popular docking programs is required to ensure a successful docking outcome. In this study, we systematically compared the performance of three popular docking programs, Autodock, Autodock Vina, and Surflex-Dock for a series of structurally similar weekly binding flavonoids (22) binding to the estrogen receptor alpha (ERα). For these flavonoids-ERα interactions, Surflex-Dock showed higher accuracy than Autodock and Autodock Vina. The hydrogen bond overweighting by Autodock and Autodock Vina led to incorrect binding results, while Surflex-Dock effectively balanced both hydrogen bond and hydrophobic interactions. Moreover, the selection of initial receptor structure is critical as it influences the docking conformations of flavonoids-ERα complexes. The flexible docking method failed to further improve the docking accuracy of the semi-flexible docking method for such chemicals. In addition, binding interaction analysis revealed that 8 residues, including Ala350, Glu353, Leu387, Arg394, Phe404, Gly521, His524, and Leu525, are the key residues in ERα-flavonoids complexes. This work provides reference for assessing molecular interactions between ERα and flavonoid-like chemicals and provides instructive information for other environmental chemicals. Display omitted •The predicted binding performance of flavonoids to ERα is evaluated.•Surflex-Dock shows higher docking accuracy than Autodock and Vina for flavonoids.•The prediction by flexible docking method is not improved for flavonoids with ERα.•The binding mechanism of flavonoids with ERα is indicated.