We report a highly sensitive fluorescent probe based on p-dimethylaminobenzoyl derivatives (probe L) for the detection of Cu2+ and Zn2+. In this work, the probe L exhibited a fluorescent turn-on sensing model to Cu2+ and Zn2+ with a distinct fluorescent color change from colorless to green and yellow respectively. Probe L exhibited high selectivity as a fluorescent Cu2+/Zn2+ probe with a limit of detection (LOD) of 45 nM/17 nM. The results of 1H NMR titrations revealed that the response of L to Cu2+ and Zn2+ was triggered by the interaction of the thiophene unit and the metal ion. Furthermore, the fluorescence titrations and Job's plot curves displayed the binding ratio of 1:2 for Cu2+ and 1:1 for Zn2+ metal-L complex formation respectively. Density functional theory calculation also demonstrated the possibility of molecular luminescence and the process of metal-L complex formation. Additionally, fluorescent test strips have been prepared for convenient detection of Cu2+ and Zn2+, which means the convenient and rapid assay in real samples can be achieved.