Non-canonical G-quadruplex (G4) DNA structures play key roles in cellular regulation and are promising targets for cancer therapy. This study reports the design, synthesis, and biophysical evaluation of 15 novel pyridine bis-quinazoline derivatives for their ability to selectively bind and stabilize G4 DNA structures. The pyridine-bis-quinazoline central fragment was synthesized with various amine side chains via a 4-5 step sequence in high yields. Comprehensive analyses using different fluorescence resonance energy transfer (FRET) assays, fluorescence intercalator displacement (FID), circular dichroism (CD), and nuclear magnetic resonance (NMR) assays revealed strong G4 stabilization and selectivity over double-stranded DNA. The presence and composition of the aliphatic amine side chain proved critical and propylamine linkers exhibited superior performance, achieving ΔTm values exceeding 20 °C and dissociation constants in the nanomolar range. Structural preferences were observed for parallel and hybrid G4 topologies, and the ligands induced minimal conformational changes in G4 DNA upon binding. Finally, cell viability assays on HCT-8 and HepG2 cancer cell lines revealed that most ligands effectively entered the cells and decreased cancer cell viability in a dose-dependent manner. These findings underline the potential of pyridine bis-quinazoline derivatives as selective G4-stabilizing agents, paving the way for further exploration in anticancer drug development.