Efficient molecular selection is a prerequisite for generating molecular tools used in diagnosis, pathology, vaccinology, and therapeutics. Selection efficiency is thermodynamically highly dependent on the dissociation equilibrium that can be reached in a single round. Extremely shifting of equilibrium towards dissociation favors the retention of high-affinity ligands over those with lower affinity, thus improving the selection efficiency. For the first time, we propose to synergize dual effects by deterministic lateral displacement microfluidics, including the collision-based force effect and the 2D separation-based concentration effect, to greatly shift the equilibrium. Compared with previous approaches, this system can remove more low- or moderate-affinity ligands and maintain most high-affinity ligands, thereby improving affinity discrimination in selection. This strategy is demonstrated on phage display in both experiment and simulation, and two peptides against tumor markers EphA2 and CD71 were obtained with high affinity and specificity within a single round of selection, which offers a promising direction for discovery of robust binding ligands for a wide range of biomedical applications.