The invention discloses a quantum dot ratio fluorescent temperature-sensitive probe based on interface defects and a preparation method of the quantum dot ratio fluorescent temperature-sensitive probe. A ZnInS/ZnS quantum dot is used as a main body material and defect fluorescence is brought through doping elements Ag and Mn; visual ratio temperature detection can be carried out by adopting changes of two fluorescent peak ratios under different temperatures, wherein the Ag&Mn: ZnInS/ZnS quantum dot ratio fluorescent probe adopts a core-shell structure in order to enhance the fluorescence. A one-pot hot injection method is adopted by the invention, has the advantages of simple process, easiness in operation, convenience for supplying raw materials and low price of the raw materials and can be finished in common chemical laboratories, so that the quantum dot ratio fluorescent temperature-sensitive probe is easy to popularize.