A direct and environmentally friendly synthesis method was developed to produce aqueous CdS quantum dots (QDs) at room temperature. The transmission electron microscopy (TEM) and X-ray diffraction (XRD) results showed the small size and the cubic zinc blende structure of the nanocrystals. The quantum yield was comparable to that of the commercial core-shell QDs. With 3-mercaptopropionic acid (MPA) as the capping molecule, the feasibility of using the aqueous CdS QDs as imaging tool was demonstrated with Salmonella typhimurium cells. The photoluminescence (PL) properties of the present aqueous CdS QDs can be optimized by adjusting various processing parameters. The emission was due to trap states and was related to the dispersion condition. In particular, with higher pH and MPA/Cd ratio of 2, the QDs exhibited stronger emission. The temperature- and concentration-dependent properties of QDs resulted from the intrinsic interactions between nanoparticles. The aqueous CdS QDs displayed long lifetime of 12 h under UV light and excellent stability in DI water, PBS, and cytosol for more than 26 days. The ease of processing and good PL properties of the aqueous CdS QDs provide a practical and economical approach for single-target imaging application.