Carbon dots (CDs) are a new class of photoluminescent (PL), biocompatible, environment-friendly, and low-cost carbon nanomaterials. Synthesis of highly efficient red-emitting carbon dots (R-CDs) on a gram scale is a great challenge at present, which heavily restricts the wide applications of CDs in the bioimaging field. Herein, R-CDs with a high quantum yield (QY) of 53% are produced on a gram scale by heating a formamide solution of citric acid and ethylenediamine. The as-prepared R-CDs have an average size of 4.1 nm and a nitrogen content of about 30%, with an excitation-independent emission at 627 nm. After detailed characterizations, such strong red fluorescence is ascribed to the contribution from the nitrogen- and oxygen related surface states and the nitrogen-derived structures in the R-CD cores. Our R-CDs show good photostability and low cytotoxicity, and thus they are excellent red fluorescence probes for bioimaging both in vitro and in vivo.