Here, we present a novel, semiautomated protein-DNA interaction characterization technology, selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq). SMiLE-seq is neither limited by DNA bait length nor biased toward strong affinity binders; it probes the DNA-binding properties of TFs over a wide affinity range in a fast and cost-effective fashion. We validated SMiLE-seq by analyzing 58 full-length human, mouse, and Drosophila TFs from distinct structural classes. All tested TFs yielded DNA-binding models with predictive power comparable to or greater than that of other in vitro assays. De novo motif discovery on all JUN-FOS heterodimers and several nuclear receptor-TF complexes provided novel insights into partner-specific heterodimer DNA-binding preferences.