Gel beads are formed when alginate acid reacts with divalent cations, particularly Ca2+. As a result of this feature, it is one of the best materials for making gel beads. Furthermore, it swells only slightly at acidic pH, resulting in stable alginate acid beads, but swells and dissolves rapidly at higher pH values, leading to pH-responsive release. Our current study aimed to embed folate-modified chitosan 5FU nanoparticles (FA-CS-5FU-NPs) into calcium alginate beads for colon-targeted delivery. Calcium alginate beads were developed successfully. Based on the method of drying, two types of beads were obtained: freeze-dried folate-modified chitosan 5FU nanoparticles-embedded beads (FA-CS-5FU-NP-Bf) and oven-dried folate-modified chitosan 5FU nanoparticles-embedded beads (FA-CS-5FU-NP-Bo). The size of (FA-CS-5FU-NP-Bf) was significantly larger than (FA-CS-5FU-NP-Bo). Swelling index (SI), erosion index (EI), and water-uptake index (WUI) of (FA-CS-5FU-NP-Bf) beads were significantly higher than FA-CS-5FU-NP-Bo beads at simulated intestinal pH. An insignificant difference was observed in the release rate of 5FU between (FA-CS-5FU-NP-Bf) and FA-CS-5FU-NP-Bo. The release rate of FA-CS-5FU-NPs was significantly higher than FA-CS-5FU-NP-Bf and FA-CS-5FU-NP-Bo. Pharmacokinetic parameters of 5FU solution, FA-CS-5FU-NPs, and FA-CS-5FU-NP-Bo were analyzed. Solution of pure 5FU showed significantly higher Cmax and lower AUC, T1/2, and Vd than both FA-CS-5FU-NPs and FA-CS-5FU-NPs-Bo, suggesting that FA-CS-5FU-NPs and FA-CS-5FU-NPs-Bo have sustained-release behavior. Biodistribution studies also show that maximum drug amounts were found in the colon from nanoparticles-embedded beads. FA-CS-5FU-NPs-Bo avoid releasing drugs in the stomach and small intestine and make them available in the colon region in higher concentrations to target the colon region specifically.