Six novel di-substituted phenanthroline derivatives 5a–7a and 3b–5b have been prepared, and their binding interactions with human telomeric (h-telo) and promoter (c-kit2 and c-myc) G-quadruplex DNAs were investigated. All the compounds are potent stabilisers of the G-quadruplex structures and compounds 3b, 4b, and 5b exhibit high G-quadruplex DNA selectivity over duplex DNA. The binding affinities of these compounds to G-quadruplex DNA are higher than to duplex DNA. CD spectra show that the compound can induce the formation of an anti-parallel structure of the h-telo G-quadruplex. Each h-telo quadruplex binds two compound molecules by the end-stacking mode. Six new compounds are able to inhibit significantly the telomerase activity at low μM concentration.