We rapidly prepared photoluminescent carbon dots (CDs) using a single-step heat reflux method with L-glutamic acid as the carbon source. The as-prepared CDs possessed a quasispherical morphology with an average diameter of approximately 5.42 nm and a quantum yield of approximately 6.3%. The CDs clearly exhibited excitation-dependent photoluminescence and good temperature-sensitive photoluminescence, as well as excellent water-solubility. Moreover, the luminescent CDs could not only be efficiently taken up by CT26.WT and CAL-27 cells but also exhibited low cytotoxicity and favourable biocompatibility, making them promising candidates for cellular imaging applications, among others.