摘要： Owing to their unique superiorities in chemical and photoluminescence (PL) stability, low toxicity, biocompatibility, and easy functionalization, graphene quantum dots (GQDs) were widely used in cell imaging, probes, and sensors. However, further development and deeper research of GQDs were restricted by their imprecise and complex structure and accompanying controversial PL mechanism. In this work, two kinds of structure defined water-soluble GQDs, with different oxidation degree, were synthesized from molecules by using bottom-up syntheses methods. After studied by a serial of characterizations, their optical properties, functional groups, molecular weight, and structural information were obtained. The optical properties of GQDs could be optimized by controlling their oxidation degree. PL mechanism of GQDs was investigated by comparing their structure and properties. Furthermore, robust, stable, and precise temperature probes were designed by using the GQDs, which exhibited an excellent wide responding range, which covered the whole physiology temperature range, from 0 ℃ to 60 ℃ in water. Moreover, the GQDs were successfully applied as temperature responsive fluorescence probe in Hela cell line. These works put forward a solid foundation for the applications of biological thermo probes and selectively temperature detectors in vitro cellular and in vivo.