Noninvasive and accurate measurement of intracellular temperature is of great significance in biology and medicine. This paper describes a safe, stable, and accurate intracellular nano-thermometer based on an l-DNA molecular beacon (l-MB), a dual-labeled hairpin oligonucleotide built from the optical isomer of naturally occurring d-DNA. Relying on the temperature-responsive hairpin structure and the FRET signaling mechanism of MBs, the fluorescence of l-MBs is quenched below the melting temperature and enhanced with increasing temperature. Because of the excellent reversibility and tunable response range, l-MBs are very suitable for temperature sensing. More importantly, the non-natural l-DNA backbone prevents the l-MBs from binding to cellular nucleic acids and proteins as well as from being digested by nucleases inside the cells, thus ensuring excellent stability and accuracy of the nano-thermometer in a complex cellular environment. The l-MB nano-thermometer was used for the photothermal study of Pd nanosheets in living cells, establishing the nano-thermometer as a useful tool for intracellular temperature measurement.