As ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs) are widely distributed in the central and peripheral nervous systems and are associated with the pathogenesis of various degenerative neurological diseases. Here, we report the results of phage display-based de novo screening of an 11-residue linear peptide (named LKP1794) that targets the α7 nAChR, which is among the most abundant nAChR subtypes in the brain. Moreover, two d-peptides were generated through mirror image and/or primary sequence inverso isomerization (termed DRKP1794 and DKP1794) and displayed improved inhibitory effects (IC50 = 0.86 and 0.35 μM, respectively) on α7 nAChR compared with the parent l-peptide LKP1794 (IC50 = 2.48 μM), which markedly enhanced serum stability. A peptide-based fluorescence probe was developed using proteolytically resistant DKP1794 to specifically image the α7 nAChR in living cells. This work provides a new peptide tool to achieve inhibitory modulation and specifically image the α7 nAChR.