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As a sustainable and ecological dye resource, biomass materials are garnering increasing attention. In this study, pigments extracted from grouper root were utilized for dyeing modified cotton fabrics, and various properties were investigated. First, the optimal parameters for extracting dyes from the grouper root using ultrasound were investigated. The extracted dyes were analyzed by UV/Vis, FTIR, and liquid chromatography–mass spectrometry. Subsequently, the extracts were applied to dye specially modified cotton fabrics, and potential mechanisms were proposed. The color fastness, UV resistance, antioxidant properties, and antibacterial efficacy were evaluated post-dyeing. The results indicated that the primary constituents of grouper pigments are anthocyanins. After modifying the cotton fabrics with 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) and chitosan, the dyeing performance of the fabrics with root extracts was significantly enhanced. Furthermore, the samples exhibited improved dyeing performance and functionality following the CHPTAC modification treatment. The dyed fabric achieved a K/S value of 9.1564, wash and rubbing fastness above 4.0, a ultraviolet protection factor of 204.17, over 80% antioxidant activity, and >95% antimicrobial efficacy against Escherichia coli and Staphylococcus aureus. Excellent performance persisted after 20 washes, demonstrating the potential of natural dyes in developing durable, functional textiles.

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