Carbon dots (CDs) with both one- and two-photon fluorescence have attracted much attention in the field of bioimaging. In this study, we reported a strategy for the design of peptide-conjugated CDs for cellular nuclear-targeted imaging. The CDs were prepared from amino acid and formic acid via an one-step hydrothermal synthesis method, and then modified with trans-activator of transcription (TAT) peptide for both one- and two- photon nuclear-targeted fluorescence imaging. The CDs derived from tryptophan (Trp) and formic acid showed the highest fluorescence quantum yield of 58.4% with an average size about 1.7 nm in diameter. The maximum excitation and emission wavelengths of the Trp/formic acid CDs were 360 and 442 nm, respectively. The Trp/formic acid CDs were successfully conjugated with TAT peptide through the reaction between the amino group of the peptide and carboxylic group of the CDs. Upon grafting with TAT peptide, the resulted TAT-Trp/formic acid CDs could be used as nuclear-targeted fluorescent probes for both one- and two-photon live cell fluorescence imaging.