Phosphorous and nitrogen co-doped carbon dots (P,N-CD) with satisfactory quantum yield have been prepared through one-step acidic oxidation of pumpkin by H 3 PO 4 at low temperature (90 o C). The asprepared P,N-CD is relatively monodisperse with a narrow size distribution. The P,N-CD displays a remarkable emission enhancement in the yellow fluorescence region (λ em = 550 nm) when the pH is 10 increased from 1.5 to 7.4. The pK a of P,N-CD was found to be 4.17 and it shows linear response to physiological range of pH 4.7-7.4, which is valuable for near-neutral cytosolic pH research. It is observed that P,N-CD is a superior fluorescent bioimaging agent in animals and cells thanks to its excellent solubility and ultra-low toxicity. In addition, P,N-CD displays a notably large Stokes shift of 125 nm, good reversibility and could effectively avoid the influence of autofluorescence in biological systems. 15 The confocal fluorescent microscopic images of subcellular distribution and the detection of pH in MCF-7 cells were achieved successfully, suggesting that P,N-CD has excellent cell membrane permeability and is further applied successfully to monitor pH fluctuations in live cells with negligible autofluorescence. 65 Recently, there has been a trend to synthesise doped CD from natural biomass as they are inexpensive, inexhaustible and nontoxic. Several successful demonstrations were given to prepare nanomaterials using biomass materials which had many potential applications. 7,[42][43][44] We are interested in developing 70 convenient ways to synthesise P,N-CD from environmentally friendly materials and simple apparatus. As we all know, pumpkin abounds with carbon, nitrogen, oxygen, phosphorus, sulfur, and hydrogen elements owing to the existence of carbohydrate, protein, lipid, and glutathione. Thus, we anticipate 75 that this natural material should be a promising precursor for