Rosa setate x Rosa rugosa is widely used in the essential oil industry and generates large amounts of waste annually. The purpose of this research is the recycling of bioactive flavonoids from rose waste biomass to develop high-value products. Resin screening and adsorption/desorption dynamic analysis showed that HP20 resin was suitable to purify the flavonoids from R. setate x R. rugosa waste extracts. Under the optimal enrichment process, the product had a 10.7-fold higher purity of flavonoids with a satisfactory recovery of 82.02%. In total, 14 flavonoids were identified in the sample after purification by UHPLC-QTOF-MS. Moreover, the DPPH and ABTS assays revealed that the flavonoids-purified extracts exhibited higher antioxidant activities than the crude extracts. Meanwhile, the purified extracts presented stronger antiproliferative activity against HepG2, Caco-2, MCF-7 and A549 cell lines. The bacteriostatic effects of the purified extracts against four bacteria (Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Staphylococcus epidermidis (S. epidermidis), Pseudomonas aeruginosa (P. aeruginosa)) and yeast (Candida albicans (C. albicans)) were stronger compared with the crude extracts. It was concluded that flavonoids-enriched extracts from R. setate x R. rugosa waste had the potential to be applied in functional food and pharmaceutical industries.