Synthesis of folic-acid mediated targeting nanocarrier is one of the most important and effective approaches in drug delivery. In this study, firstly, the surface of polyamidoamine G4 (PAMAM G4) dendrimers was modified with polyethylene glycol (PEG) to reduce dendrimer toxicity and was engineered with folic acid (FA) to increase nanocomplex targeting of the mice myoblast C2C12 cell lines. Both these synthetic phases were characterized via fourier transform infrared spectroscopy (FT-IR), Proton nuclear magnetic resonance (H-1 NMR), ultraviolet-visible spectrophotometry (UV-Vis), and differential scanning calorimetry (DSC) analysis. Secondly, 5-fluorouracil (5-FU), as a chemotherapeutic agent, was loaded in the nanocarrier to therapeutic goals and was characterized. In vitro drug release was also assessed and an excellent controlled release with good release efficiency was reported. The size, diameter, surface morphology, and surface charge of these synthesized nanocomplexes were ascertained with the scanning electron microscopy (SEM), dynamic light scattering (DLS) and zeta potential analyser, respectively, and PAMAM G4 positive charges eliminated. Finally, our nanocomplex was tested on the HT-29 human colorectal cancer and mice myoblast C2C12 normal cell lines and cell viability was evaluated with an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Good cancer cell inhibition and cell growth promotion effects of the nanocomplexes have been indicated in this research study.