Biopanning refers to the processes of screening peptides with a high affinity to a target material. Microfluidic biopanning has advantages compared to conventional biopanning which requires large amounts of the target material and involves inefficient multiple pipetting steps to remove nonspecific or low-affinity peptides. Here, we fabricate a microfluidic biopanning system to identify a new gold-binding peptide (GBP). A polydimethylsiloxane microfluidic device is fabricated and bonded to a glass slide with a gold pattern that is deposited by electron-beam evaporation. The microfluidic biopanning system can provide high adjustability in the washing step during the biopanning process because the liquid flow rate and the resulting shear stress can be precisely controlled. The surface plasmon resonance analysis shows that the binding affinity of the identified GBP is comparable to previously reported GBPs. Moreover, molecular dynamics simulations are performed to understand its binding affinity against the gold surface in detail. Theoretical calculations suggest that the association and dissociation rates of the GBPs depend on their sequence-dependent conformations and interactions with the gold surface. These findings provide insight into designing efficient biopanning tools and peptides with a high affinity for various target materials.