Abstract: This review summarized recent advances in thermosensitive fluorescent nanomaterials for biomedical applications, focusing on their use in intracellular temperature monitoring, tumor-microenvironment sensing, and the construction of multifunctional theranostic platforms. These nanomaterials exhibited fluorescence changes in response to external stimuli, offering non-invasive, real-time feedback with high spatiotemporal resolution. Temperature variations could be reported through multiple optical parameters such as emission intensity and peak position. For intracellular thermometry, they enabled real-time tracking of temperature fluctuations within living cells, providing crucial insights into cellular physiology. In tumor-microenvironment monitoring, they detected localized temperature alterations, facilitating early diagnosis and guiding therapy. Under photothermal treatment, the nanomaterials were delivered to tumor sites and, upon laser irradiation, generated localized hyperthermia to selectively ablate cancer cells. Nevertheless, clinical translation still faced challenges, including long-term biosafety evaluation, stability in complex physiological environments, and optimization of response accuracy. Future research would concentrate on developing materials with higher biocompatibility and intelligent responsiveness, integrating multimodal sensing and multifunctional capabilities, achieving subcellular-organelle targeting for precision therapy, promoting synergistic tumor-specific hyperthermia and chemotherapy, and ultimately constructing “all-in-one” theranostic platforms.