Forkhead box protein 1 (FoxA1) is a member of the forkhead family of winged helix transcription factors that plays pivotal roles in the development and differentiation of multiple organs and in the regulation of estrogen-stimulated genes. Conventional analytical methods - electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy (FA) - as well as a gold nanoparticles (AuNPs)-based assay were used to study DNA binding properties of FoxA1 and ligand interruption of FoxA1-DNA binding. In the AuNPs assay, the distinct ability of protein-DNA complex to protect AuNPs against salt-induced aggregation was exploited to screen sequence selectivity and determine the binding affinity constant based on AuNPs color change and absorbance spectrum shift. Both conventional EMSA and FA and the AuNPs assay suggested that FoxA1 binds to DNA in a core sequence-dependent manner and the flanking sequence also played a role to influence the affinity. The EMSA and AuNPs were found to be more sensitive than FA in differentiation of sequence-dependent affinity. With the addition of a spin filtration step, AuNPs assay has been extended for studying small molecular ligand inhibition of FoxA1-DNA interactions enabling drug screening. The results correlate very well with those obtained using FA.