Abstract We report the use of carrot, a new and inexpensive biomaterial source, for preparing high quality carbon dots (CDs) instead of semi-conductive quantum dots for bioimaging application. The as-derived CDs possessing down and up-conversion photoluminescence features were obtained from carrot juice by commonly used hydrothermal treatment. The corresponding physiochemical and optical properties were investigated by electron microscopy, fluorescent spectrometry, and other spectroscopic methods. The surfaces of obtained CDs were highly covered with hydroxyl groups and nitrogen groups without further modification. The quantum yield of as-obtained CDs was as high as 5.16%. The cell viability of HaCaT cells against a purified CD aqueous solution was higher than 85% even at higher concentration (700 μg mL−1) after 24 h incubation. Finally, CD cultured cells exhibited distinguished blue, green, and red colors, respectively, during in vitro imaging when excited by three wavelength lasers under a confocal microscope. Offering excellent optical properties, biocompatibility, low cytotoxicity, and good cellular imaging capability, the carrot juice derived CDs are a promising candidate for biomedical applications.