A novel flow injection (FI) on-line displacement/sorption preconcentration and separation technique coupled with FAAS was developed for the determination of trace copper in complicated matrices. The methodology involved on-line formation of lead diethyldithiocarbamate (Pb–DDTC), pre-sorption of the resultant Pb–DDTC onto the inner walls of the knotted reactor (KR), retention of the analyte CuII on the inner walls of the KR through a displacement reaction between CuII and the sorbed Pb-DDTC and elution of the retained analyte with ethanol for FAAS detection. Interference from co-existing ions with lower stability DDTC complexes relative to Pb-DDTC were eliminated without need for any masking reagents in the proposed on-line displacement/sorption preconcentration system. The developed method was shown to tolerate up to ≥250 mg l−1 FeIII, 40 mg l−1 CdII, ≥30000 mg l−1 ZnII, ≥25000 mg l−1 MnII and 10000 mg l−1 CoII in the sample digest. With 70 s of displacement/sorption at a flow rate of 6.0 ml min−1, an enhancement factor of 155 and a detection limit (3s) of 0.38 µg l−1 were obtained. The precision (RSD, n = 11) was 0.65% at the 50 µg l−1 level and 3.3% at the 5 µg l−1 level. The developed system was successfully applied to interference-free determination of trace copper in a variety of environmental and biological materials with high contents of co-existing heavy metals.