Abstract: Phenanthroline (PNT) photospectrometry is often used for the determination of Fe(II), due to Fe(III) in the mixture interferes with the Fe(II) test system this method cannot simultaneously determine Fe(II) and Fe(III). So, a computational PNT photospectrometry for the simutaneous determination of Fe(II) and Fe(III) in bioleaching samples has been carried out. The linear effect of Fe(III) to the Fe(II) test system was first evaluated, and the interference of the typical metal ions (Cu²⁺, Ni²⁺, Cd²⁺, Co²⁺) in the bioleachates and dissolution of samples were investigated. Using this method, Fe(II) and Fe(III) of the typical secondary iron minerals and in the leachates can be determined accurately. It is easier and fast for the simultaneous analysis of Fe(II) and Fe(III) in the actual bioleaching residues and cell surface.