The 9‐ and 12‐dimethylaminophenyl‐substituted berberine derivatives 3 a and 3 b were readily synthesized by Suzuki‐Miyaura reactions and shown to be useful fluorescent probes for the optical detection of quadruplex DNA (G4‐DNA). Their association with the nucleic acids was investigated by spectrometric titrations, CD and LD spectroscopy, and with DNA‐melting analysis. Both ligands bind to duplex DNA by intercalation and to G4‐DNA by terminal π stacking. At neutral conditions, they bind with higher affinity (K b=105−106 M−1) to representative quadruplex forming oligonucleotides 22AG, c‐myc, c‐kit, and a2, than to duplex calf thymus (ct) DNA (K b=5‐7×104 M−1). At pH 5, however, the affinity of 3 a towards G4‐DNA 22AG is higher (K b=1.2×106 M−1), whereas the binding constant towards ct DNA is lower (K b=3.9×103 M−1) than under neutral conditions. Notably, the association of the ligand with DNA results in characteristic changes of the absorption and emission properties under specific conditions, which may be used for optical DNA detection. Other than the parent berberine, the ligands do not show a noticeable increase of their very low intrinsic emission intensity upon association with DNA at neutral conditions. In contrast, a fluorescence light‐up effect was observed upon association to duplex (Φ fl=0.01) and quadruplex DNA (Φ fl=0.04) at pH 5. This fluorimetric response to G4‐DNA association in combination with the distinct, red‐shifted absorption under these conditions provides a simple and conclusive optical detection of G4‐DNA at lower pH.