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An inproved phenanthroline (PNT) photospectrometry for simutaneous determination of Fe(Ⅱ) and Fe(Ⅲ) in bioleaching samples was studied.Specificity analysis indicated in the iron mixtures the total Fe content can be quantitatively determined by the total iron test system, and the Fe(Ⅱ) content determination by the Fe(Ⅱ) test system is affected by the presence of Fe(Ⅲ), and such an influence increases linearly with Fe(Ⅲ) content.Based on which, a computational spectrophotometry with the modified linear equations were set up.