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Alcohol has been demonstrated to disrupt intestinal barrier integrity. Some flavonoid compounds that exert antioxidant activity have a protective effect on intestinal barrier function. As an important medicinal and edible plant, sea buckthorn (<i>Hippophae</i>) seeds are rich in flavonoids, but their protective effect on the intestinal barrier has not been reported. In our research, 76 kinds of flavonoids were identified in <i>Hippophae rhamnoides</i> ssp. <i>sinensis</i> seed residue flavonoids (HRSF) by ultra-performance liquid chromatography–tandem mass spectrometry. Kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, kaempferol-3-O-robinoside-7-O-rhamnoside, isorhamnetin-3-O-2G-rhamnosylrutinoside, quercetin-3-O-rutinoside, (−)-epigallocatechin, and B type of procyanidin were the most abundant substances, accounting for 15.276%, 15.128%, 18.328%, 10.904%, 4.596%, 5.082%, and 10.079% of all identified flavonoids, respectively. Meanwhile, pre-treatment with HRSF was able to prevent alcohol-induced disruption of intestinal barrier integrity through elevating the transepithelial monolayer resistance value, inhibiting the flux of fluorescein isothiocyanate-dextran, and upregulating the mRNA and protein level of TJs (occludin and ZO-1). Furthermore, it was also able to reverse alcohol-induced oxidative stress through suppressing the accumulation of reactive oxygen species and malondialdehyde, improving the glutathione level and superoxide dismutase activity. Finally, the results showed that HRSF pre-treatment effectively elevated the erythroid-related factor 2 mRNA and protein level compared with the alcohol-alone treatment group. Our research was the first to demonstrate that HRSF could prevent alcohol-induced intestinal barrier dysfunction through regulating the Nrf2-mediated pathway in order to attenuate oxidative stress and enhance TJ expression.