In order to study the differences of dissolved organic matter (DOM) characteristics between the secondary effluent and the surface water, ultrafiltration (UF) was combined with three-dimensional excitation-emission matrix (EEM) spectroscopy to investigate the fluorescence characteristics of total DOM and different molecular weight fractions of them. Moreover, the DOM removal characteristics by coagulation were analyzed also under the respective optimal dosage of coagulant (PAC). The results showed that: DOM of secondary effluent contained four notable fluorescence peaks: humic acid-, fulvic acid-, aromatic protein- and soluble microbial product-like fluorophores, which were more than surface water. Coagulation was easy to remove macromolecules as humus, but the removal efficiency was not high enough for the relative small molecules as protein and microbial products. Fluorescence index (FI) indicated that secondary effluent after biotreatment were mainly from microbial sources. The correlation relationship between the humus-, protein-like fluorescence intensity and UV254 are strong in secondary effluent.