In the current work, we investigated and compared the capability of the monodispersed chitosan (CS) microspheres, CS coated zinc-based metal-organic framework (CS/Zn-MOF) microspheres, and CS coated the hybrid of Zn-MOF with graphene oxide (CS/Zn-MOF@GO) microspheres as an anticancer drug carrier. The prepared samples were characterized through the common characterization techniques. The morphologies, sizes, and drug loading efficiency of the prepared compounds respectively were well-characterized, obtained, and compared with each other. The scanning electron microscopy (SEM) analysis displayed the particle size of the prepared microspheres in the range of 20–40 µm. The highest amount of 5-fluorouracil (5-Fu) loading was found for the ternary hybrid of CS/Zn-MOF@GO microspheres; ~45%. The 5-Fu loaded CS microspheres (5-Fu@CS/Zn-MOF@GO microspheres) with rough surfaces exhibited a pH-sensitive and sustained release pattern for loaded 5-Fu. So, the cumulative drug release amount at pH 5.0 was relatively two times greater than pH 7.4. Eventually, CS/Zn-MOF@GO microspheres exhibited acceptable enzymatic biodegradability and also good biocompatibility toward MDA-MB 231 cells line as an epithelial, human breast cancer cell. 41.2% cell viability after 48 treatment with 5-Fu@CS/Zn-MOF@GO microspheres confirmed the capability of 5-Fu loaded CS/Zn-MOF@GO microspheres in the tumor cell treatment. Based on the founded interesting results the CS/Zn-MOF@GO ternary hybrid microspheres could be proposed as a promising and hopeful candidate to applied in the localized anticancer delivery.