Binding of nuclear proteins to sequence-specific regulatory elements on chromosome is the fundamental importance in gene expression and Epigenome. DNA pulldown assay uses a biotinylated double stranded DNA probe to selectively extract protein-DNA complexes from cell nuclear extract. Analysis of the eluted protein complexes from DNA probe using mass spectrometry or western blot subsequently is an important step in elucidating the mechanisms by which gene expression are regulated. However, with the current DNA pulldown protocol, results always demonstrate significant nonspecific bindings and high backgrounds. Optimising the assay with multiple different binding conditions might be laboriously and economically challenging. Here, we have developed a rapid and sensitive 384-well fluorescence polarisation assay to screen multiple 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 light is emitted in the same polarized 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 controls. We applied this method to demonstrate a repertoire of protein complexes recruited to Sox2/Oct4 binding motif upstream of F-box protein 15 gene (Fbxo15) in mouse embryonic stem cell. Our preliminary results showed that this method should contribute to a better understanding of different proteins associated with a specific DNA sequence in vitro.