The regulation of hydrophilicity/hydrophobicity of carbon dots (CDs) at will is most important. One pot simultaneous preparation of hydrophilic and/or hydrophobic CDs is herein reported, via a hydrothermal process with 1-butyl-3-methylimidazolium hexafluorophosphate as the carbon source in a H3PO4-ethanol medium. The hydrophilicity or hydrophobicity of CDs (or their proportions) is simply regulated by varying the H3PO4/ethanol molar ratio. Hydrophilic and hydrophobic CDs are obtained simultaneously with H3PO4/ethanol molar ratios within 0-1.72, while hydrophilic or hydrophobic CDs are the sole product obtained from H3PO4-BmimPF6 or BmimPF6-only systems. The CDs exhibit excitation-dependent maximum fluorescence at 360/440 nm (hydrophilic) and 430/510 nm (hydrophobic), with quantum yields of 17.0% and 7.7%, respectively. Both hydrophilic and hydrophobic CDs obtained by this approach exhibit favorable biocompatibility and offer great potential in bio-imaging as demonstrated for the fluorescent labeling and imaging of live HeLa cells.