A novel fluorescent probe (TPA-TF) was designed and synthesized, incorporating triphenylamine-thiophene moieties coupled with furan and a hydrazone. First, the TPA-TF can be used as a sensor to detect Cu2+ in the environment/biosystem through an effective fluorescence quenching due to chelation-enhanced fluorescence quenching (CHEQ) and inhibiting the photoinduced electron transfer (PET) process. Various fluorescence spectra and density functional theory (DFT) computations were used to clarify the TPA-TF to Cu2+ detection characteristics. The Cu2+ sensor TPA-TF exhibited a fast response time of one minute. In this study, the linear range concentration for the Cu2+ ion detection was 0-1000 μM, with a broad pH working range (pH 2.7–7.4), and the detection limit of the sensor was 6.08 μM. With its broad applicability, the TPA-TF sensor was successfully used to detect and photograph Cu2+ in live cells, demonstrating its considerable potential for applications in biological imaging.