Copper ion (Cu2+) and l-cysteine (l-Cys) detection is critically important since an abnormal level of Cu2+ or l-Cys is an indicator for many diseases. In this paper, we demonstrate an “off–on” approach for highly sensitive and selective detection of Cu2+ and l-Cys using carbon dots (CDs) as fluorescent probes. CDs were prepared by using mesoporous silica (MS) spheres as nanoreactors. The binding ability of CDs towards metal ions was examined by comparing the fluorescence intensities of CDs before and after the addition of the metal ions. The addition of Cu2+ cations leads to their absorption on the surface of CDs and the significant fluorescence quench of CDs (turn-off). The resulting in CDs-Cu2+ system was found to be sensitive to l-Cys. The addition of l-Cys not only serves to shelter the CDs effectively from being quenched, but also to reverse the quenching and restore the fluorescence (turn-on) due to its ability to remove Cu2+ from the surface of CDs. This method is facile, rapid, low cost, and environment-friendly. A detection limit as low as 2.3×10−8 M for Cu2+ and 3.4×10−10 M for l-Cys is obtained, which is promising for biological applications.