The Cu(2+)-imprinted cross-linked chitosan resin was synthesized and the binding characteristic of the resin to Cu(2+) was evaluated. The prepared resin was packed into a micro-glass column and used as micro-separating column. The micro-separating column was connected into the chemiluminescence flow system and placed in front of the window of the photomultiplier tube. Based on the luminol-hydrogen peroxide chemiluminescence system, a flow injection online chemiluminescence method for determination of trace copper was developed and trace Cu(2+) in complex samples was successfully determined. The proposed method improved the shortcomings of chemiluminescence method's poor selectivity.