Pesticide residue contamination has become an urgent issue since it threatens both the natural environment and public health. In this study, a fluorescent method for detecting dithiocarbamate (DTC) compounds was constructed based on novel nickel nanoclusters (Ni NCs) and copper ions (Cu). The water-soluble fluorescent Ni NCs were synthesized for the first time through a one-pot method using glutathione as stabilizer and ascorbic acid as reducing agent. The as-prepared Ni NCs exhibited a maximum fluorescence emission at 445 nm when excited by 380 nm. And they displayed aggregation-induced emission enhancement when ethylene glycol was introduced into the nanocluster aqueous solution. Based on the Ni NCs, a label-free fluorescence quenching sensor was established for sensitive and selective detection of DTC compounds with the assistance of Cu. The complex formed by DTC and Cu led to fluorescence quenching of Ni NCs through inner filter effect. The sensing method was successfully applied to two typical DTC compounds, thiram and disulfiram, with good linearity over a wide linear range and a low detection limit. Moreover, the proposed approach was capable of thiram detection in real samples, which confirms the potential of this sensing method as a platform for DTC compound detection.