A highly sensitive and accurate dual-signal strategy is developed for trace Cu2+detection based on the inner filter effect (IFE) between Tween 20-gold nanoparticles (AuNPs) and CdSe/ZnS quantum dots (QDs). Tween 20-AuNPs are utilized as colorimetric probes and excellent fluorescent absorbers. The fluorescence of CdSe/ZnS QDs can be quenched efficiently by Tween 20-AuNPs via IFE. In the presence of D-penicillamine, D-penicillamine induces the aggregation of Tween 20-AuNPs and the fluorescent recovery of CdSe/ZnS QDs at high ionic strength. Upon addition of Cu2+, D-penicillamine tends to selectively chelate with Cu2+and then forms the mixed-valence complexes, which consequently inhibits the aggregation of Tween 20-AuNPs and the fluorescent recovery. The dual-signal method is used to quantitatively detect trace Cu2+, with low detection limits of 0.57 μg/L and 0.36 μg/L for colorimetry and fluorescence, respectively. In addition, the proposed method using a portable spectrometer is applied to the detection of Cu2+in water. This sensitive, accurate and miniature sensing system has potential in environmental evaluations.