A new in-syringe membrane SPE and solid-phase visible spectral method was proposed for the rapid extraction and visible spectral determination of trace Cu2+. The chelation and membrane SPE can be accomplished in a syringe. The yellow Cu(DDTC)2 complex was separated using a polyethersulfone membrane from the sample solution. Then, the complex can be detected directly on the polyethersulfone membrane utilizing solid-phase visible absorbance spectra without elution. The proposed method simplified the experimental procedure and improved the sensitivity to the μg L-1 level. Furthermore, this method is environmentally friendly since it avoids the use of organic solvents. After the investigation of the influence of different variables on the membrane SPE procedure, water and blood plasma were analyzed to validate the proposed method. A LOD of 0.04 μg L-1 and recoveries of 96.0-103.7% confirmed that the present work can be applied for the determination of trace Cu2+ in water and blood plasma samples.