Objective To investigate the trends in enzyme activity and structural changes during black tea processing, and to analyze the functional expression of polyphenol oxidase (PPO), peroxidase (POD), β-glucosidase (β-GC), and protease, aiming to optimize the processing technology of Gougunao black tea.Methods Using one bud and one leaf of the Gougunao population as raw materials, Gougunao black tea was prepared by traditional processing technology. The relative browning degree and tea polyphenol content during processing were measured. The activity and structural changes of PPO, POD, β-GC, and protease were analyzed, and the enzyme functional expression mechanisms were explored.Results The relative browning degree increased during black tea processing, reaching a maximum of 503.21% during the first drying stage. Tea polyphenol content decreased to 15.4%, with the largest decline occurring after fermentation. Activities of PPO, POD, and β-GC showed a decreasing trend, whereas protease activity exhibited an increase-decrease-increase pattern. UV-visible and Fourier transform infrared (FTIR) spectra revealed significant conformational changes in PPO during fresh leaves, withering, and rolling. POD's enzyme structure changed during fermentation, first drying, and final drying, with a marked decrease in activity during fermentation. Beta-GC exhibited distinct absorption peaks during withering, rolling, and drying, with a continuous decrease in activity. Protease showed characteristic absorption peaks throughout processing, with activity decreasing during withering and increasing during first drying. X-ray diffraction (XRD) spectra indicated that PPO, β-GC, and protease were amorphous polymers, while POD's three-dimensional conformation changed.Conclusion Significant changes occurred in the enzyme activities and structures of PPO, POD, β-GC, and protease during Gougunao black tea processing.