Verification of the existence of quadruplex structure in native human telomeres and determination of the major structure of d(T2 AG3 )4 (H24) in K+ solution are the major questions regarding the structure of human telomeres. We have synthesized a fluorescent probe of 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) that has a very high binding affinity for G-quadruplex H24. BMVC stabilizes quadruplex structures and acts as a sensitive probe to the local environment. Although the circular dichroism patterns of H24 are different in Na+ and K+ solutions, similar binding behaviors of BMVC to H24 in these solutions led us to suggest that the major G-quadruplex structure of H24 in K+ solution is very likely similar to that in Na+ solution. Of particular interest is the fluorescent band detected at −575 nm in quadruplex H24 and at −545 nm in duplex DNA. In addition, the intensity of BMVC fluorescence increases by two orders of magnitudes upon interaction with either duplex or G-quadruplex DNA. BMVC has a greater binding preference for G-quadruplex H24 than for duplex DNA. Analyzing the BMVC fluorescence at the ends of metaphase chromosomes and other regions of chromosomes allowed us to verify the presence of G-quadruplex structure in human telomeres for the first time. Using fluorescence lifetime imaging microscopy, the longer decay time of BMVC in G-quadruplex H24 than in duplex DNA allowed us to map the G-quadruplex structure in human metaphase chromosomes.