Since its discovery, graphene has become a prominent material of interest for advanced bioelectronic and biomedical applications such as diagnostics, drug delivery, and imaging. This two-dimensional atomically thin sheet of sp2 hybridized carbon has exceptional electronic properties and is well suited for the development of highly sensitive and selective biosensors when paired with biomolecular adlayers. Additionally, by controlling the biomolecular orientation, conformation, and assembly structure of the adlayer, device functionality and performance can be fine-tuned. In this work, we focus on the conformational properties of three graphene-binding peptides, GrBP5-WT, GrBP5-M2 and Truncated GrBP5-M2, that form strongly adhered self-assembled adlayers at graphene surfaces. While these peptides are chemically very similar, experimental observations revealed they demonstrate opposite assembly phenomena upon thermal stimulus. Herein, enhanced sampling molecular dynamics simulations were employed using GROMACS simulation package with the PLUMED plugin to unravel how specific peptide conformations lead to the unexpected assembly behavior. Self-assembling peptide conformations were identified by comparing their computationally derived binding energies to experimental energetic data obtained from a scanning probe microscopy based molecular energetic analysis. The self-assembly structure of peptide on the graphene surface could form a module on the biosensor and used to detect specific proteins or peptides. With a better understanding of the peptide self-assembly mechanism, it would be significant to the development of biosensor.