Binding nuclear proteins to sequence-specific regulatory elements on chromosomes is fundamental to regulating gene expression and the epigenome. The standard DNA pulldown assay uses a biotinylated double-stranded DNA probe to selectively extract protein-DNA complexes from cell nuclear extracts. The analysis of the eluted protein complexes from the DNA probe using mass spectrometry or western blot is an essential step in elucidating the mechanisms by which gene expression is regulated. However, with the current DNA pulldown protocol, the results have consistently demonstrated significant, nonspecific binding and high amounts of background noise. Optimising the assay with multiple different binding conditions is laboriously and economically challenging. Here, we have developed a rapid and sensitive 384-well fluorescence polarisation assay to screen various binding conditions for DNA pulldown. This assay is based on the principle that when proteins bind to the fluorescence-labelled DNA probe, the fluorescent molecules become stationary, and the light emitted is in the same polarised plane as the excitation light. This method has been shown to be useful in determining the optimal protein binding conditions for sequence-specific DNA probes and reduced nonspecific bindings for scrambled controls. We applied this method to demonstrate a repertoire of protein complexes recruited to the Sox2/Oct4 binding motif upstream of the F-box protein 15 gene (Fbxo15) in mouse embryonic stem cells. Our preliminary results showed that this method contributes to a better understanding different proteins associated with a specific DNA sequence in vitro.