Both G-quadruplex (G4) and G-triplex (G3) can bind thioflavin T (ThT) to light up the fluorescence of ThT. G4/ThT and G3/ThT can be used as fluorescent indicators to construct a label-free molecular beacon (MB). In this work, we present a comparative perspective of G3/ThT-based MB and G4/ThT-based MB. The results showed that the G3/ThT-based MB had higher sensitivity and faster response speed than the G4/ThT-based MB. Furthermore, we systematically studied the effect of stem length and varying pairs on the response of the G3/ThT-based MB, and then proposed one rational design of the G3/ThT-based MB. This work demonstrates that the shorter G3 is more suitable for constructing the MB stem. This present work opens a promising way to develop a sensitive, simple and homogeneous biosensing method.