Biopanning, a common affinity selection approach in phage display, has evolved numerous ligands for diagnosis, imaging, delivery, and therapy applications. However, traditional biopanning has suffered from time-consuming processes, highly-repetitive procedures and labor-intensive manual operation. Herein, a highly integrated and automated biopanning platform (Auto-Panning) is proposed. Based on digital microfluidics (DMF), biopanning processes are integrated on a chip with highly reproducible, precise, automated liquid manipulations. Therefore, 3 rounds of Auto-Panning can be accomplished within 16 h, instead of nearly a week of complicated manual operations. Auto-Panning has been used to evolve a specific peptide against cancer biomarker EphA2 with excellent cellular penetrating ability and significant invasion suppression biofunction, successfully demonstrating the practicality of the platform. Overall, as an automated programmable molecular screening platform, Auto-Panning will further promote the discovery and applications of novel ligands.