In this study, a novel fluorescent probe based on CuInS2 quantum dots modified with alanine (Ala-CuInS2 QDs) was developed for the detection of lead ions and cysteine (Pb2+ and Cys). Ala-CuInS2 QDs were synthesized through a one-step hydrothermal method exhibiting uniform size, good stability and water solubility. The QDs were then utilized as an "on-off-on" fluorescence sensor to detect Pb2+ and Cys in the ranges of 0-20 µM and 0-55 µM respectively, with detection limits of 0.29 µM and 0.66 µM. The mechanism of fluorescence quenching and recovery processes was also explored. Furthermore, Ala-CuInS2 QDs have been successfully applied to detect Pb2+ in tap and river water and detect cysteine in serum.