Straw incorporation is significant for straw reduction and reutilization, and is clearly required in the twelfth five-year-plan for national economic and social development of the People’s Republic of China. The incorporated straw will naturally decompose and release the component of dissolved organic matter (DOM). At present, it lacks the research on straw humification behavior controlled by environmental factors and complexation effect between humification component and metal ions with fluorescence spectrometry in the representative region of loess. The fluorescence spectrometry was used to reveal the 3D-EEMs characteristics of DOM affected by temperatures and lead ions in the straw humification process, and the modified Stern-Volmer equation and Van’t Hoff equation were applied to indicate the complexation parameters and thermodynamic constants between lead ions and DOM. The results showed: the humification temperatures affected little on fluorescence peaks of DOM and no peaks were obviously found to appear or disappear from the 3D-EEMs results. The fluorescence intensity decreased gradually at higher temperatures and in the presence of lead ions, the quenching effect might work in the process. The binding ability was more significant between lead ions and visible fulvic-like component shown from modified Stern-Volmer equation, and the values of f revealed the complexation effect of lead ions and functional groups in DOM. Static quenching was the primary mechanism during the reaction process. The constants in Van’t Hoff equation suggested the reaction was spontaneous and endothermic, and the disordered degree and the complexity were relatively low in the reaction system. The 3D-EEMs were acceptable to illustrate the variation of DOM characteristics under different temperatures and in the presence of lead ions in the straw humification process.