The central loop of G-quadruplex molecular beacons is a key element to sense target DNA or RNA sequences. In this study, circular dichroism spectroscopy (CD), thermal difference spectrum (TDS), non-denatured non-denaturing gel electrophoresis, and thermal stability analysis were used to investigate the effect of the central loop length on G-quadruplex features. Two series of G-quadruplexes, AG₃TTAG₃-(TTA)n-G₃TTAG₃T (n = 1–8) (named TTA series) and AG₃TTTG₃-(TTA)n-G₃TTTG₃T (n = 1–8) (named TTT series) were examined in K⁺ and Na⁺ solutions, respectively. CD and TDS spectral data indicated that TTA series adopted an antiparallel G-quadruplex structure in Na⁺ solution and a hybrid G-quadruplex structure in K⁺ solution respectively. TTT series exhibited a hybrid G-quadruplex structure in both Na⁺ and K⁺ solutions. UV melting curves indicated that the stability of G-quadruplex in both series was reduced by the elongation of central loop. Thermal stability analysis concluded that the G-quadruplex destabilization with long central loop is an entropy-driven process due to more flexible and longer central loops.