In the last two decades, near-infrared (NIR) emitting Cu-doped quantum dots (QDs) have stimulated stupendous research interest for their excellent optical properties. Due to the synthetic method in the organic phase, almost none of the NIR emitting Cu-doped QDs were used as the fluorescent probes for biological imaging, despite being employed for applications in many fields. In this work, we successfully synthesized water-soluble Cu-doped CdS quantum dots (Cu:CdS d-dots) with an emission wavelength at 722 nm using a fast and facile hydrothermal method. The optical properties of the d-dots and several key parameters of the synthesis conditions such as dopant concentration, reaction temperature, reflux time, and especially the pH of the reaction mixture were systematically studied, and the mechanism of these parameters is also discussed. A red shift of the emission maximum as a consequence of the increased reaction temperature was observed, but it does not move to the NIR region until Cu 2+ is doped even at the highest reflux temperature in aqueous solution. The d-dots have good water solubility and biocompatibility for successful labeling the living HeLa cells as fluorescence probe.