Abstract: The conversion and utilization of biomass resources are of great significance in environmental protection. In this study, nitrogen-doped yak hair biomass composite carbon dots (N-CDs) were successfully prepared through steam flash pyrolysis and hydrothermal methods using yak hair solid waste as the carbon source. With antibacterial activity as the evaluation criterion, the preparation parameters were optimized using response surface methodology, resulting in carbon dot composites with excellent antibacterial performance, achieving a 96.8% inhibition rate against Escherichia coli. The morphology, structure, and optical properties of the prepared N-CDs were systematically investigated using TEM, XPS, FT-IR, UV-Vis, and FL spectroscopy. Characterization results showed that the particle size of N-CDs significantly decreased (1.5 ± 0.4 nm) compared to nitrogen-doped carbon dots before doping (3.5 ± 0.8 nm). The introduction of nitrogen led to increased oxygen and nitrogen content, particularly quaternary ammonium groups, while the optimal excitation and emission wavelengths exhibited a red shift. These structural, functional group, and optical property changes collectively enhanced the antibacterial activity of N-CDs. Antibacterial experiments demonstrated that the optimized N-CDs exhibited significant inhibitory effects on both Escherichia coli and Staphylococcus aureus. At a concentration of 0.8 mg/mL (MIC), bacterial colony growth and formation were almost undetectable. The prepared N-CDs were added to a gelatin-based solution for strawberry film-coating preservation, which effectively delayed strawberry spoilage, significantly reduced weight loss and bacterial proliferation, and maintained the fruit's nutritional value. This study provides a green strategy for developing highly efficient antibacterial carbon dots derived from natural biomass and demonstrates promising application potential in the field of agricultural product preservation.