Biomolecular detection and imaging methods provide quantitative measurements essential for biological research. In this context, molecular beacon based sensors have emerged as powerful, no-wash imaging agents, providing target-specific fluorescent activation for nucleic acids, proteins, and small molecules. Conventional molecular beacons require double-labeled DNA sequences, which are costly and time-consuming to prepare. To address this issue, we developed DNA based label-free molecular beacons consisting of two regions: a signal-generating region based on human telomeric G-quadruplex sequence that activates Thioflavin T fluorescence and a target recognition sequence designed to interact in a molecular beacon format. We demonstrated the utility of these probes for the selective detection of DNA, RNA, and protein. Multiple probes were applied against a single target to achieve improved brightness in fluorescence detection of nucleic acid targets. This label-free strategy provides a straightforward, cost-effective alternative to fluorescently labeled oligonucleotides in biomolecular detection and imaging.