Fluorescein can emit strong and stable fluorescence. Cu2+ can oxidize fluorescein, which causes the fluorescence signal to diminish. Cu(DP)2+ (DP refers to α,α′‐dipyridyl) and Cu‐GPD‐4.0 (GPD‐4.0 refers to 4.0‐generations polyamidoamine dendrimers) both can catalyze Cu2+ to oxidize fluorescein, which causes the fluorescence signal to diminish sharply. The ΔF is directly proportional to the content of copper. Based on the facts above, a new catalytic fluorescence spectrophotometry for the determination of trace copper using Cu(DP)2+ and Cu‐GPD‐4.0 was established. The linear range of this method is 0.040–28 pg mL−1. The regression equation for working curve is ΔF=209.5+14.39 CCu 2+ (pg mL−1), n=7; correlation coefficient is 0.991. The detection limit of this method is 1.0×10−14 g mL−1. After replicate measurement times, RSDs are 3.1% and 4.2% for samples containing 0.040 and 28 pg mL−1 Cu2+, respectively. This method is rapid and precise with high sensitivity and good repeatability. The method has been applied to the determination of trace copper in tea and human hair with satisfactory results. Meanwhile, the mechanism for the determination of trace copper by catalytic fluorescence spectrophotometry using Cu(DP)2+ and Cu‐GPD‐4.0 was also discussed.