Precise description of temperature at the microscale level is essential in many biological applications. In this study, we prepared a DNA-based thermometer that reports low and high temperatures by providing two distinct optical signals. The system is a molecular beacon that carries a loop and a stem, whose conformation is subject to change from a hairpin to a random coil when the temperature changes from low to high. A fluorophore, Cy5, and a quencher, BHQ3, are terminally labeled at the stem ends. Moreover, perylene is included in the middle of the 3'-end stem. The signaling state of Cy5 relies on the relative distance to BHQ3. However, the perylene emission is regulated by its microenvironment (i.e., the oligonucleotide or duplex state). With a temperature variation, the designed thermometer undergoes a change in conformation that leads to two signal patterns with Cy5/off and perylene/on at low temperature and Cy5/on and perylene/off at high temperature. The reversibility and biocompatibility of the thermometer design were examined for potential applications in biological systems.