Carbon dots (CDs) have outstanding optical properties, biocompatibility, and photostability, making them attractive for imaging applications. A facile and green one-step hydrothermal synthesis method is proposed, which can be safely used in a wide range of applications such as chemical sensing, bioimaging, and optoelectronics. In this study, we report green synthesis of carbon dots from bitter orange juice (Citrus Aurantium) by hydrothermal treatment for the first time. We studied effects of time, temperature, and pH on fluorescence of CDs, characterized them using various spectroscopic and microscopic methods, and evaluated their toxicity to different cell lines. Identifying an optimum reaction condition of 180 ºC for 7 h heating gave CDs that showed pH-dependent fluorescence, with the largest fluorescence at a pH of 7.0. The CDs were 1-2 nm in size with a spherical morphology and negative surface charge. The CDs showed a high quantum yield of 19.9 %, reasonable photostability, excellent water solubility, and long fluorescence lifetime. A one step hydrothermal rout led to various hydrophilic functional groups on the surface of the CDs. Our results showed that the CDs were non-toxic over a large concentration range and effective for imaging of cells, indicating their potential as imaging probes in medical diagnostics and biosensor applications.