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Abstract Efficient red emissive carbon dots (CDs) in aqueous solutions are very scarce for high performance bioimaging applications. In this work, we report a one-step solvothermal treatment to synthesize pure red emissive CDs (FA-CDs) from citric acid and urea in formic acid without complicated purification procedures. Photoluminescence quantum yield (PLQY) of 43.4% was observed in their dimethyl sulfoxide solutions. High PLQY up to 21.9% in aqueous solutions was achieved in their bovine serum albumin (BSA) composites (FA-CDs@BSA) with significantly enhanced multi-photon fluorescence. The strong surface electron-withdrawing structure of FA-CDs caused by the high content of C = O groups contributes for their pure red emission. Owing to the significantly enhanced single and multi-photon red fluorescence and enlarged particle sizes after composing with BSA, in vivo tumor imaging and two-photon fluorescence imaging of blood vessels in mouse ear have been realized via intravenous injection of FA-CDs@BSA aqueous solutions.