G-quadruplex oligonucleotides (GQs) exhibit specific anti-proliferative activity in human cancer cell lines, and they can selectively inhibit the viability/proliferation of cancer cell lines vs. non-cancer ones. This ability could be translated into a cancer treatment, in particular for glioblastoma multiform (GBM), which currently has a poor prognosis and low-efficiency therapeutic treatments. A novel bi-modular GQ, bi-(AID-1-T), a twin of the previously described three-quartet AID-1-T, was designed and studied in terms of both its structure and function. A covalent conjugation of two AID-1-Ts via three thymidine link, TTT, did not interfere with its initial GQ structure. A comparison of bi-(AID-1-T) with its mono-modular AID-1-T, mono-modular two-quartet HD1, and bi-modular bi-HD1, as well as conventional two-quartet AS1411, was made. Among the five GQs studied, bi-(AID-1-T) had the highest anti-proliferative activity for the neural cancer cell line U87, while not affecting the control cell line, human embryonic fibroblasts. GQs, for the first time, were tested on several primary glioma cultures from patient surgical samples. It turned out that the sensitivity of the patient primary glioma cultures toward GQs varied, with an apparent IC50 of less than 1 μM for bi-(AID-1-T) toward the most sensitive G11 cell culture (glioma, Grade III).