The 3' human telomeric overhang provides ample opportunities for the formation and interaction of G-quadruplexes, which have shown impacts on many biological functions including telomerase activities in the telomere region. However, in the few investigations on DNA constructs that approach to the full length of the human telomeric overhang, the presence of higher-order quadruplex-quadruplex interactions is still a subject of debate. Herein, we employed dynamic splint ligation (DSL) to prepare a DNA construct, 5'-(TTAGGG)24 or 24G, which has the length comparable to the full stretch of 3' human telomeric overhang. Using mechanical unfolding assays in laser tweezers, we observed a minor population (∼5%) of higher-order interactions between G-quadruplexes, while the majority of the quadruplexes follow the bead-on-a-string model. Analyses on the noninteracting G-quadruplexes in the 24G construct showed features similar to those of the stand-alone G-quadruplexes in the 5'-(TTAGGG)4 (4G) construct. As each 24G construct contains as many as six G-quadruplexes, this method offers increased throughput for the time-consuming mechanical unfolding experiments of non-B DNA structures.