Abstract: Tremella fuciformis polysaccharides (TFPS) are natural substances with important biological activities, but their activities are easily affected by coexisting proteins. Therefore, effective removal of proteins is a key step to enhance the bioactivity of TFPS. This study aimed to screen out the most suitable method for removing proteins from polysaccharides to enhance the structural stability and biological activity. Taking the protein removal rate and polysaccharides retention rate as evaluation indicators, this study systematically compared the effects of three deproteinization methods, namely the Sevage method, trichloroacetic acid (TCA) method, and hydrochloric acid (HCl) method, on Tremella fuciformis polysaccharides. The polysaccharides obtained by these methods were analyzed for their structural characteristics using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and high-performance liquid chromatography. The antioxidant activities of the polysaccharides were comprehensively evaluated based on their free radical scavenging abilities. The experimental results showed that the Sevage method achieved a protein removal rate of 75.47% and a polysaccharides retention rate of 60.94% after four treatments. When the TCA concentration was 20%, the protein removal rate was 24.84%, and the polysaccharides retention rate was relatively high (83.78%). The HCl method at pH2 yielded a protein removal rate of 32.64% and a polysaccharides retention rate of 65.60%. Structural analysis demonstrated that the Fourier-transform infrared (FTIR) spectra of polysaccharides subjected to distinct deproteinization treatments exhibited no significant disparities, with the same functional group absorptions. However, marked differences were observed in their molecular weight distributions. The main molecular weight of the Sevage-treated polysaccharides was approximately 900 kDa, with fewer low-molecular-weight components. In contrast, those obtained through the trichloroacetic acid (TCA) method displayed a multi-modal and broad molecular weight distribution. Polysaccharides obtained by the HCl method showed a broad molecular weight range, from 40 kDa to 2000 kDa. Scanning electron microscopy (SEM) analysis further revealed that the sheet-like structure of the polysaccharides became thinner following deproteinization. The results of antioxidant activity determination indicated that the polysaccharides obtained by the Sevage method had significantly higher free radical scavenging ability than those obtained by the other two methods. At the highest concentration of 2.5 mg/mL, the scavenging rates of DPPH, hydroxyl and ABTS⁺ free radicals reached 70.56%, 29.51% and 37.44%, respectively. In conclusion, different deproteinization methods significantly influenced the structural characteristics and antioxidant activities of Tremella fuciformis polysaccharides. Among them, the Sevage method provided the most effective protein removal and yielded polysaccharides with stronger bioactivity, making it a preferable approach for the purification and functional development of Tremella fuciformis polysaccharides.