Among the most common cancers, colon cancer is the fourth. To develop new methods with controlled-release manner, more attention was devoted to designing efficient oral delivery systems. For this purpose, 5-fluorouracil (5-FU) as an anticancer drug was encapsulated into the porous of Zn-based metal-organic framework (MOF-5) through immersing in the drug solution. pH-sensitive carboxymethylcellulose (CMC) biopolymer was used to protect and carry the 5-FU encapsulated MOF-5 nano-hybrid (5-FU@MOF-5) through the digestive system. The CMC-coated 5-FU@MOF-5 bio-nanocomposite hydrogel bead (CMC/5-FU@MOF-5) characterized using various techniques. To demonstrate the efficiency of CMC/5-FU@MOF-5 as a novel carrier for controlled release of 5-FU, in-vitro drug delivery tests were carried out in simulating the gastrointestinal tract (GIT) conditions. The drug release tests revealed that the CMC/5-FU@MOF-5 has a sustained release manner in the GIT conditions. The MTT test established that the CMC/5-FU@MOF-5 has notable toxicity against HeLa cells. According to the obtained results, the prepared bio-nanocomposite hydrogel beads could be proposed as a potential drug delivery system for the colonic administration of 5-FU.