We successfully developed three simple yet novel one-pot synthetic approaches for the carbon dots (CDs): (1) The oligonucleotides served as a “biosolvent” to enable direct extraction and modification of CDs. (2) A cigarettes-assisted apparatus facilitated one-pot preparation of both hydrophilic and hydrophobic CDs. Finally, (3) the size- and color-tunable CDs that emit wavelengths from blue to red were achieved by using a single precursor in pure water. These fluorescent CDs were stable for several months under ambient conditions and show negligible photobleaching under continuous laser irradiation at a wide range of pH. The low toxicity, biocompatibility and high quantum yield of CDs render the potential for in vitro and in vivo bioimaging and future drug delivery applications.