In this study, a trial aims to decrease side effects and increase the efficacy of 5FU was established through active targeting of folate receptor overexpressing cancer cells using bovine serum albumin as a nanocarrier and folic acid as a targeting ligand. Physical characterization of size, potential, and polydispersity index (PDI) was performed, and (265.45 nm) particles with (- 31.1 mV) potential and (0.072) PDI were produced. For evaluation of the efficacy of the suggested formulation against the free form of the 5FU, a cytotoxic assay was performed on (Caco2, MCF7), a human colon cancer cell line, and a human breast cancer cell line, respectively, with different folate receptor-expressing features. MTT, NR & ATP assays were used, and all showed higher significant efficacy and a lower side effect of the formulation. Biochemical analysis of oxidative stress biomarker {Malonaldehyde (MDA)}, antioxidants biomarkers {Glutathione reductase (GR), Glutathione peroxidase (GPx)}, and a detoxifying enzyme {Glutathione transferase (GST)} was carried out along with quantitative real-time polymerase chain reaction (PCR) analysis of B cell lymphoma 2 (Bcl-2) gene. The results confirmed the cytotoxicity assay results. These results give new hope for using 5FU in a more effective and safe way in treatment.