A variety of nanostructured diagnostic tools have been developed for the precise detection of known genetic variants. Molecular beacon systems are very promising tools due to their specific selectivity coupled with relatively lower cost and time requirements than existing molecular detection tools such as next generation sequencing or real-time PCR (polymerase chain reaction). However, they are prone to errors induced by secondary structure responses to environmental fluctuations, such as temperature and pH. Herein, we report a temperature-insensitive, bead-immobilized, molecular beacon-equipped novel DNA nanostructure for detection of cancer miRNA variants with the consideration of thermodynamics. This system consists of three parts: a molecular beacon for cancer-specific RNA capture, a stem body as a core template, and a single bead for solid-support. This DNA system was selectively bound to nanosized beads using avidin–biotin chemistry. Synthetic DNA nanostructures, designed based on the principle of fluorescence-resonance enhanced transfer, were effectively applied for in vitro cancer-specific RNA detection. Several parameters were optimized for higher performance, with a focus on thermodynamic stability. Theoretical issues regarding the secondary structure of a single molecular beacon and its combinatory forms were also studied. This study provides design guidelines for new sensing systems of miRNA variation for next-generation biotechnological applications.