Strong fluorescence and low cytotoxicity ZnSe/ZnS quantum dots (QDs) were synthesized by a facile aqueous phase route. It overcame the defects such as instability and low quantum yield of the quantum dots synthesized by early aqueous phase route. L-Glutathione (GSH) and 3-mercaptopropaonic acid (MPA) were used as mixture stabilizers to synthesize high quality ZnSe/ZnS QDs. The samples were characterized by X-ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectrometry (XPS) and their optical properties were investigated by using UV-vis spectrophotometer, fluorescence spectrophotometer (FL), IR spectrophotometer and confocal laser scanning microscope. The synthesized ZnSe/ZnS QDs illuminated blue fluorescence under ultraviolet lamp. Its water-soluble property is excellent and the fluorescence intensity of ZnSe/ZnS QDs almost did not change after 4 months at room temperature. The average diameter of ZnSe/ZnS nanocrystals is about 3 nm and quantum yield (QY) could reach to 70.6% after repeat determination. Low cytotoxicity was ensured by investigated SCG7901 and RAW264.7 cells. In comparison with cadmium based nanocrystals, ZnSe/ZnS QDs posed low cytotoxicity. The cells viability remained 96.7% when the QDs concentration was increased to 10 μmol/L. The results in vitro indicate that ZnSe/ZnS QDs-based probes have good stability, low toxicity and biocompatibility for fluorescence imaging in cancer model system.