To investigate the effects of acetylation modification on the biological activities of Dendrobium huoshanense polysaccharides (DHP), acetylated D. huoshanense polysaccharides (A-DHP) were prepared using the acetic anhydride method, and the preparation process was optimized. The degree of substitution was used as the evaluation index, and synthesis conditions were optimized using single-factor experiments and response surface methodology. The structure of A-DHP was characterized using Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). Changes in antioxidant and antitumor activities before and after acetylation were evaluated using in vitro antioxidant essays and cytotoxicity experiments. The results showed that the optimal preparation conditions were a reaction time of 3.8 h, an acetic anhydride-to-polysaccharide ratio of 14:1 (mL:g), and a reaction temperature of 36 ℃. Under these conditions, the degree of substitution of A-DHP reached 0.226. FTIR and SEM analyses confirmed the successful introduction of acetyl groups into the DHP structure. In vitro antioxidant assays demonstrated that acetylation increased the scavenging activities against ABTS and DPPH free radicals by 14.91% and 7.76%, respectively. In addition, cytotoxicity assays showed that the inhibitory effects of A-DHP on HepG2 and C918 cells increased by 15.90% and 16.24%, respectively. Overall, acetylation modification effectively enhanced the antioxidant and antitumor activities of DHP. These findings provide a theoretical basis for the further development and application of acetylated polysaccharides.