G-triplexes have recently been identified as a new kind of DNA structures. They perhaps possess specific biological and chemical functions similar as identified G-quadruplex but can be formed by shorter G-rich sequences with only three G-tracts. However, until now, limited G-triplexes sequences have been reported, which might be due to the fact that their stability is one of the biggest concerns during their functional studies and application research. Herein, we found a G-rich sequence (5'-TGGGTAGGGCGGG-3') which can form a stable G-triplex (Tm ∼ 60 °C) at room temperature. The stable G-triplex can combine with thioflavin T and function as an efficient fluorescence light-up probe. Comparing with the traditional G-quadruplex based probe, this triplex based probe was easy to be controlled and excited. Finally, the probe was successfully applied into constructing a label-free molecular beacon for miRNA detection. Taking advantage of these abilities of the G-triplex based fluorescent probe, the challenges faced during designing G-rich sequences based fluorescent biosensors can be efficiently solved. These findings provide important information for the future application of G-triplex.