Carbon dots (CDs) are a new class of photoluminescent nanomaterials with high photostability, good biocompatibility in contrast to the traditional quantum dots. The wavelength-tuneable emission property and one- and two-photon emission ability have made them promising candidates as new ‘nanolights’ for biological applications. Herein, we report the design and synthesis of CDs by using citric acid and urea as the carbon sources via a facile hydrothermal strategy. The quantum yield of the CDs can reach as high as 29%. FTIR spectra show that there are amino and carboxylic groups on the surface of CDs, which can be used for further surface modification with functional moieties. After conjugation with triphosphonium (TPP), the size of the CDs changes from 12 nm to 20 nm. The TPP-CDs show maximum emission wavelength at 416 and 448 nm under the excitation of 340 nm (one-photon emission) and 800 nm (two-photon emission), respectively. The two-photon emission was further confirmed by the quadratic relationship of the fluorescence intensity with excitation laser power. TPP-CDs exhibit good pH stability in the range of pH 2 to 11 and excellent photostability against photo-bleaching as compared with fluorescein. Confocal microscopy analysis demonstrated that the TCA-8113 tongue squamous carcinoma cells incubated with TPP-CDs resulted in greater uptake of TPP-CDs in the mitochondria by using MitoTracker as a control. Similar results were also observed in B16-F10 mouse melanoma cells. Quantitative analysis revealed that a Pearson’s correlation coefficient of 0.63 can be achieved, suggesting a good colocalization of the TPP-CDs with MitoTracker in the mitochondria. These results demonstrated that the TPP-CDs may have the ability for both one- and two-photon mitochondrial-specific live cell imaging.