Rapid and non-invasive temperature detection inside biological tissues can be realized through a fluorescence intensity ratio thermometry technology, but it is still restricted due to the thermal quenching of fluorescence and the large absorption and scattering coefficients of tissues in practical applications. In this paper, cubic phase ScF3:Yb3+/Er3+ nanocrystals with negative thermal expansion characteristics were synthesized by a one-step hydrothermal method, in which the thermal enhancement of up-conversion (UC) luminescence was achieved due to the thermally improved energy transfer efficiency from a shortened distance between the activator and sensitizer. For further realizing its application in biology, Nd3+ was introduced in ScF3:Yb3+/Er3+ to construct a high-sensitivity temperature probe with near-infrared (NIR) excitation and NIR emission. Furthermore, calibrating the temperature measurement curve and designing the ex vivo experiment rationally can give a potential temperature detection of the present sample inside the tissue. The results indicate a strategy for solving the UC luminescence thermal quenching and developing NIR emission temperature probes for organisms.