<p>Polysaccharide-based drug delivery systems have gained significant attention due to their biocompatibility and potential for targeted therapy. In this study, we utilized <i>Ganoderma lucidum</i> polysaccharides (GLP) as a macromolecular carrier to synthesize polysaccharide drug conjugates by forming ester bonds with 5-fluorouracil (5-FU) derivatives (5-FUAC). This not only improves the solubility and stability of 5-FU but also enhances its targeted delivery to tumor tissues, potentially reducing systemic toxicity. Utilizing <i>Ganoderma lucidum</i> polysaccharides (GLP) as a macromolecular carrier, we successfully synthesized GLP-5-FU conjugates by forming ester bonds with 5-fluorouracil derivatives (5-FUAC). These conjugates were then characterized using spectrum analysis technologies. The drug loading capacities of 5-FU in the GLP-5-FU conjugates were found to be approximately 4.7%. Morphological observations revealed that the GLP-5-FU conjugates had a regular spherical shape. In terms of drug release, 5-FU was found to be released faster at pH 6.8 compared to pH 7.4. To investigate the dynamic viscoelastic behavior of GLP and GLP-5-FU conjugates in water, dynamic rheology analysis was conducted. In terms of cytotoxicity, the GLP-5-FU conjugates exhibited greater inhibition of proliferation compared to 5-FU and GLP alone in HeLa, 786-O, and SKOV3 cells. Furthermore, the GLP-5-FU conjugates showed lower cytotoxicity to 293A cells.</p> Graphical Abstract <p></p>