The aim of this study was to optimize the integrated SPE-FIA (Solid phase extraction-flow-injection analysis) hyphenated technique for determination of trace level of Cu(II) ions where flow-injection sample processing was used. Firstly, Humic acid (HA) was immobilized onto aminopropyl silica (APS). Then, the material was filled into the column using slurry packing procedure. The prepared column was assembled to HPLC using two valves system for SPE-FIA analysis and used for the removal of Cu(II) ions from aqueous solution. The analysis of Cu (II) ions was realized by both SPE and FIA techniques on modified HPLC system as online. In SPE system, Cu(II) ions were preconcentrated on micro column loaded with immobilized humic acid (ImHA). EDTA (pH = 8) solution was used for the elution of Cu(II) ions sorbed by ImHA. The sorption characteristics of Cu(II)-immobilized humic acid system were investigated at different Cu(II) concentrations, and output was observed by a UV detector. The developed method was applied for online metal ion determination in samples, especially Cu (II) and obtained results compared with atomic absorption spectrophotometer (AAS) results. Sorption characteristics were evaluated by using Freundlich, Langmuir, and Dubinin-Radushkevich (D-R) adsorption isotherms, as well as by Scatchard plot analysis. (C) 2009 Elsevier B.V. All rights reserved.