Heat stroke is one of the most serious causes of mortality. To prevent the situation, it is fundamental to research the mechanism of heat cytotoxicity. The preliminary results revealed that heat stroke and the change of lysosome acidity had some certain correlation. To further clarify their relationship, herein, we report a highly selective and sensitive fluorescence probe (NT1) for turn-on sensing of the pH value. NT2 was synthesized as control compound. Compared to NT2, NT1 showed accurate lysosome target ability. In addition, the suitable pKa value (5.67) allows NT1 to response to the changes of lysosomal pH values. Most importantly, NT1 could be used to study the correlation between the change of lysosomal pH and heat stroke. It was shown that the lysosomal pH value increasing with temperature during heat stroke. Thus, NT1 was an excellent candidate for research of the complex biological mechanism of heat stroke.