Previous studies have shown that the combination of biological and ozone oxidation processes can achieve a greater performance in treating natural surface water than each process individually. In this work, we designed and tested an ozonation–gravity-driven up-flow slow rate (0.01 m/h) biofiltration–ozonation (O3–GUSB–O3) process for the pre-treatment of reclaimed water prior to ultrafiltration (UF), with the aim of producing high quality drinking water and a significantly reduced degree of UF fouling. Results showed that O3 coupled with GUSB can effectively remove aromatic compounds (∼ 84.8%), dissolved organic carbon (DOC, ∼ 83.4%), and biopolymers in surface water. In addition, post-ozonation greatly contributed to the reduction of the UF membrane fouling (∼ 6 times greater flux). With regard to the disinfection by-product formation potential (DBPFP) of the final treated water, both trihalomethane formation potential (THMFP) and haloacetic acid formation potential (HAAFP) were greatly reduced (86.4% and 84.8% for THMs and HAAs, respectively). The relationship between DBPFP and various spectral indexes revealed that aromatic compounds and amino acids were more likely to generate DBPs during the disinfection stage. Among these, humic substances were more likely to generate THMs, while low molecular weight carboxylate and carbonyl organic compounds were associated with the generation of HAAs. Moreover, the dosage of O3 during the post-ozonation stage was found to influence directly the generation of DBPs. Overall, this study has conducted a detailed evaluation of a novel multi-ozone biofilter UF process for treating surface water, and the results provide a valuable basis for subsequent studies at larger scale to demonstrate the potential of the treatment process for practical applications.