The effect of metal ions and surfactants on the enzymatic hydrolysis of pretreated wheat straw lignocellulose was investigated. Scanning electron microscopy, infrared spectrum analysis, dynamic light scattering, and fluorescence spectra analysis were used to characterize the influence of Fe3+/ polyoxyethylene (20) sorbitan monooleate (Tween 80). The interaction between Fe3+/Tween 80 and enzyme was further investigated by enzyme kinetics and enzyme activity measurements. The best synergistic effect was obtained when the ratio of Fe3+ and Tween 80 was 0.06. The scanning electron microscopy images showed that the Fe3+/Tween 80 combination was associated with high porosity substrates. The infrared spectrum analysis indicated that the components of the substrates depended on additive types. The highest relative enzymatic activity increase rate was obtained with added Fe3+/ Tween 80. The Vmax and Km values of the group with added Fe3+/Tween 80 were much higher than that of the group without additives. With the addition of Fe3+/ Tween 80, the intensity of the fluorescence emission peak decreased and the peak shifted towards a longer wavelength.