The absorbances of different concentrations of Fe2+, Fe3+ and H2O2 were investigated by UV-visible spectrophotometry without separating the substances. The law of complex formation was studied by considering changes in the UV-vis spectra of mixtures of these three substances. The results show that upon eliminating the influence of the substrate, an iron-based complex was present in the Fenton reaction, which exhibited substantial absorbance from 190 to 500 nm. Therefore, the presence of an unknown complex in the Fenton oxidation process was verified and its concentration varied with a change in the concentration of hydrogen peroxide. This study provides a strong foundation for further studies into the mechanism of traditional hydroxyl radical theory of the Fenton reaction.