Cell adhesive and other functional peptides (such as RGD, KRSR, YIGSR, VAPG, and BMP-2 peptides) are extensively studied and utilized in tissue engineering scaffolds and biomedical devices to modulate cell functions. Though PEG is frequently used as the antifouling layer, it is unclear how it affects the performance of functional peptides. By analyzing the impact of PEG at short (OEG4), medium (OEG8), and long chain length (PEG2K), we reveal that PEG chain length is critical and a medium-length PEG enables functional peptides to display their optimal and genuine functions in cell adhesion, migration, and differentiation by providing excellent antifouling to minimize background noise of unwanted cell adhesion and high enough surface density of functional peptides. Our result provides new avenues for maximizing the genuine functions of peptides. This study also provides a solution to prevent the heterogeneous and even divergent results caused by inappropriate choice of antifouling PEG and provides a general guidance in identifying new functional peptides.