A rational design strategy has been developed for the construction of stable peptide-based anchors for the efficient modification of cell surfaces. Six types of peptide composed of five residues with divalent hydrophobic groups have been designed using this new strategy. Among them, a peptide with a sequence of NBD-Lys-Lys(X)-Lys-Lys-Lys(X)-NH2 (NBD: fluorophore, Lys(X): N-ε-palmitoyl-l-lysine) was found to show the highest modification efficacy and longevity in culture medium. The good performance of this peptide was attributed to (1) its high aqueous solubility, which allowed it to partition from the medium to the cell surface, and (2) the high binding affinity of the saturated palmitoyl groups to the cell membrane. We found that the distribution of the peptide was affected by recycling endosome, which enabled the representation of the peptide following its endocytotic disappearance from the cell membrane. Biotin was also presented on the cell surface using this peptide-based anchor to examine its recognition by streptavidin. The efficacy of the recognition process increased as the length of the oligoethylene glycol spacer increased, indicating that it was necessary for the biotin tag to move away from the membrane glycoproteins on the cell surface to facilitate its efficient recognition by streptavidin.