Copper ions and cysteine play a crucial role in pathophysiologic processes in the human body and the environment, so the development of probes with high sensitivity and specificity for continuous detection has become the focus of current research. In the paper, a novel fluorescent compound (named PCTMF) is synthesized with a carbazolyl thiophene group as a fluorescent moiety and formyl hydrazine as a recognition group of Cu2+. In the given DMSO-H2O solvent system, the PCTMF exhibits a highly selective and sensitive turn-off fluorescence response towards Cu2+ with its detection limit (LOD) of 0.14 μM, in the presence of other competing metal ions. The turn-off response mechanism of the probe can be attributed to the formation of complex 2PCTMF-Cu. The 2PCTMF-Cu complex presents good sensing capacity on cysteine (Cys) with a LOD of 52 nM. The molecule designing method provides an “off-on” strategy for sequentially fluorescence detecting metal ions and ligands related.