Purpose <p>Colorectal cancer remains a significant concern, demanding innovative strategies for enhanced therapeutic outcomes. This study explores the potential of nanobodies as an EGFR-targeting ligand which were conjugated to the surface of 5-fluorouracil (5-FU)-containing solid lipid nanoparticles (SLNs) in treatment of colorectal cancer.</p> Methods <p>The 7D12 nanobody was successfully expressed in <i>E. coli</i> and was further purified. The SLNs were prepared and were statistically optimized using central composite response surface methodology. Then after, the purified nanobody was chemically attached to the surface of the nanoparticles and following lyophilization, their morphology determined using scanning electron microscopy (SEM). Then, the in vitro release of 5-FU from the targeted nanoparticles was examined and finally the cytotoxicity of the nanoparticles was evaluated in SW480 and HepG2 cells.</p> Results <p>The size, poly dispersity index (PdI), zeta potential, entrapment efficiency (EE%) and loading efficiency of the optimized nanoparticles were 169.8 ± 37.17&#xa0;nm, 0.247 ± 0.062, -13.2 ± 2.18 mV, 82.9 ± 5.39% and 9.3 ± 1.06%, respectively. Although a burst release of 5-FU from nanoparticles were demonstrated in the first hours of incubation, but in further time intervals, the in vitro release profile revealed sustained release behavior. The cytotoxicity studies revealed a significant higher toxicity of targeted 5-FU-containing nanoparticles on SW480 as an EGFR- positive colorectal cancer cells in comparison with HepG2 as an EGFR-negative cell line.</p> Conclusion <p>This study paves the way for the development of an efficient, EGFR-targeted drug delivery system, with potential applications in treatment of colorectal cancer.</p>