Abstract Bacillus thuringiensis israelensis loaded on Dowex optipore V-493 as new adsorbent for the separation–preconcentration of heavy metal ions has been proposed. The analytical conditions for the quantitative recoveries of copper(II), iron(III) and zinc(II) including pH, amounts of adsorbent, sample volume, etc. were investigated. The influences of alkaline and earth alkaline ions were also reported. The recovery values for the analytes are generally higher than 95%. The preconcentration factor was 37. The limit of detections of the analyte ions ( k = 3, N = 21) were 1.14 μg L −1 for copper, 2.01 μg L −1 for iron and 0.14 μg L −1 for zinc. The relative standard deviations of the determinations were found to be lower than 9%. The procedure was validated by analyzing copper, iron and zinc contents in two certified reference materials, NRCC-SLRS-4 Riverine water and NIST SRM 1515 Apple leaves. Agreements between the obtained results and the certified values were achieved. The developed preconcentration method was applied in the flame atomic absorption spectrometric determination of copper, iron and zinc in several samples including a multivitamin–multimineral tablet, dialysis solutions, natural waters and some food samples.