Stabilization of G-quadruplexes (G4s) formed in guanine-rich (G-rich) nucleic acids by small-molecule ligands has been extensively explored as a therapeutic approach for diseases such as cancer. Finding ligands with sufficient affinity and specificity toward G4s remains a challenge, and many ligands reported seemed to compromise between the two features. To cope with this challenge, we focused on targeting a particular type of G4s, i.e., the G-vacancy-bearing G-quadruplexes (GVBQs), by taking a structure complementation strategy to enhance both affinity and selectivity. In this approach, a G-quadruplex-binding peptide RHAU23 is guided toward a GVBQ by a guanine moiety covalently linked to the peptide. The filling-in of the vacancy in a GVBQ by the guanine ensures an exclusive recognition of GVBQ Moreover, the synergy between the RHAU23 and the guanine dramatically improves both the affinity toward and stabilization of the GVBQ Targeting a GVBQin DNA by this bifunctional peptide strongly suppresses in vitro replication. This study demonstrates a novel and promising alternative targeting strategy to a distinctive panel of G4s that are as abundant as the canonical ones in the human genome.