Targeted drug delivery has been evolving at an increasing rate due to its potential to reduce the minimum effective dose of a drug and its accompanying side effects. It has shown improved therapeutic efficacy at equivalent plasma concentrations; however, the development of effective targeted delivery systems has remained a major task. In this study, a drug carrier was designed and synthesized by conjugation of folate acid (FA) to carboxymethyl chitosan (CMCS) through a polyethylene glycol (PEG) spacer. The resulting conjugates were confirmed by 1H nuclear magnetic resonance and infrared spectroscopy. The cytotoxicity of CMCS and CMCS‑5‑fluorouracil (5‑FU) was determined by a crystal violet stain assay. The potential of CMCS‑PEG‑FA for use in the targeted delivery of 5‑FU was investigated using 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide analysis in two cell lines, HeLa and A549, which contain different numbers of folate receptors on their surfaces. The MTT results revealed that in HeLa cells, the cytotoxicity of (CMCS‑5‑FU)‑PEG‑FU cells is greater compared with CMCS‑5‑FU, suggesting that folate receptor‑mediated endocytosis may affect the cellular uptake efficiency of 5‑FU‑loaded CMCS‑PEG‑FA. The CMCS‑PEG‑FA conjugates presented in this study show promise as carriers for chemotherapeutic agents due to their solubility at physiological pH, efficiency in carrying chemotherapeutic agents, low cytotoxicity and targeting ability.