A novel peptide‐based chemical fluorescence sensor L (Dansyl‐His‐Pro‐Thr‐Cys‐NH2) was designed and synthesized. This sensor exhibits an “On–Off–On” detection cycle to detect Cu2+, Zn2+, and S2− in solution. According to the chelation‐enhanced fluorescence (CHEF) mechanism, when Zn2+ is present, the fluorescence is significantly enhanced and a blue shift occurs, representing a “Turn‐On” phase of the fluorescence detection mode. Because copper ions (Cu2+) have a paramagnetic quenching sensing mechanism, the fluorescence of L quenches rapidly with the formation of the L‐Cu system, representing the “Turn‐Off” phase. The subsequent introduction of S2− to the L‐Cu system results in the recovery of the L‐fluorescence, thereby representing the second “Turn‐On” phase. As a peptide molecule, the sensor L has several advantages over other types of sensors, including water solubility, high sensitivity, and good biocompatibility, with a very low detection limit. The detection lines of Zn2+ and Cu2+ are 97 nM (R = 0.993) and 75 nM (R = 0.995), respectively. Additionally, the sensor does not exhibit any obvious cell toxicity. These results indicate that this peptide chemiluminescent sensor has the potential to be applied in in vivo detection.