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Nanofiltration (NF) is a promising post-treatment technology for providing high-quality drinking water. However, membrane fouling remains a challenge to long-term NF in providing high-quality drinking water. Herein, we found that coupling pre-treatments (sand filtration (SF) and ozone–biological activated carbon (O3-BAC)) and NF is a potent tactic against membrane fouling while achieving high-quality drinking water. The pilot results showed that using SF+O3-BAC pre-treated water as the feed water resulted in a lower but a slowly rising transmembrane pressure (TMP) in NF post-treatment, whereas an opposite observation was found when using SF pre-treated water as the feed water. High-performance size-exclusion chromatography (HPSEC) and three-dimensional excitation–emission matrix (3D-EEM) fluorescence spectroscopy determined that the O3-BAC process changed the characteristic of dissolved organic matter (DOM), probably by removing the DOM of lower apparent molecular weight (LMW) and decreasing the biodegradability of water. Moreover, amino acids and tyrosine-like substances which were significantly related to medium and small molecule organics were found as the key foulants to membrane fouling. In addition, the accumulation of powdered activated carbon in O3-BAC pre-treated water on the membrane surface could be the key reason protecting the NF membrane from fouling.