Molecular tools for double or multimeric G-quadruplexes have recently received high attention due to the possibility to increase detection sensitivity, thermal stabilization and cell imaging opportunities, in oder to determine roles and functions of G-quadruplex aggregation in medical context. In this study, two smart berberine-bisquinolinium conjugates (Ber-360A and Ber-PDS), obtained by linking the berberine fluorophore to established G-quadruplex binders (with bisquinolinium scaffold), have been synthesized and evaluated for their G-quadrulex affinity, as well as to establish mechanisms of aggregation. The conjugates are two highly selective, sensitive and fluorescent sensors able to distinguish human telomere double G-quadruplexes from other type of G-quadruplexes and ds DNA. These ligands are the first example of fluorescent tools that bind to two adjacent G-quadruplex units and optically recognize double structures. Furthermore, microscopy experiments demonstrate that the conjugate Ber-PDS enters the nucleoli and targets G-quadruplex DNA in these organelles, by simultaneously displaying a strong telomerase inhibition and antitumor activity. Two smart berberine-bisquinolinium conjugates are highly selective, sensitive and fluorescent sensors which can distinguish human telomere double G-quadruplexes from other type of G-quadruplexes and ds DNA. Conjugate Ber-PDS accumulates into the nucleoli and targets G-quadruplex DNA, by display also strong telomerase inhibition and antitumor activity. Display omitted •Two highly selective optical probes for double G-quadruplexes (G2T1) were developed.•The probes specifically target two adjacent G-quadruplex units in one G2T1.•Probe Ber-PDS allows efficient G-quadruplexes visualization by cellular microscopy experiments.•The two probes display strong telomerase inhibition and anticancer activities.