Synthesis of CuInS-based quantum dots (QDs) in the organic phase is currently one of the fastest growing points of nanotechnology. Most of the reported CuInS-based QDs are hydrophobic and cannot be directly used for biomedical applications without phase transfer. We put forward a one-pot synthetic strategy aimed at fabricating hydrophilic Zn–Cu–In–S/ZnS (ZCIS/ZnS) quantum dots (QDs) to be used directly for in vivo imaging without surface treatment. This strategy is based on the use of a hydrophilic ligand (6-sulfanyl-1-hexanol, MPH) and non-coordinating solvents such as a low molecular weight polyethylene glycol (PEG, MW = 400 Da). The capping ligand MPH endows the obtained ZCIS/ZnS QDs with good hydrophilicity and therefore offers great opportunity for direct bioimaging applications without phase transfer. Experimental results have indicated that these so-called hydrophilic ZCIS/ZnS QDs show low cytotoxicity and are successfully utilized for in vivo imaging. Furthermore, the here reported strategy doesn't only present a synthetic idea, but also might stimulate other innovative ideas on fabricating hydrophilic or water-soluble ZCIS/ZnS QDs to be used directly for biological applications.