Multi-walled carbon nanotubes (MWNTs) are suitable for delivering large biomolecules with lower cytotoxicity values and low prime cost. Surface modifications of MWNTs affect interaction with cells and proteins. Oxidation with strong acids decreases cytotoxicity of CNTs and increases protein-loading capacity. Here, after oxidation, TEM images revealed more aligned structure and carboxylated groups at the surface which decreases toxicity. Functionalized MWNTs showed more gradual degradation than the pristine MWNTs and mass loss increased by 2% in the same temperature range. Raman spectroscopy corrected graphitic structure with characteristic D and G bands at 1330 and 1579 cm−1 and increased intensity after oxidation. FTIR spectroscopy peaks at 1443 cm−1, 1560, 1640 cm−1, 2100–2200 cm−1 and 3426 cm−1 are ascribed to C–O–C vibrational stretch, C=C bonds, vibration of C≡C bonds and stretch of hydroxyl groups, respectively. The sonicationdriven dispersion of in phosphate-buffered saline, distilled water and cell culture medium were detected by UV–vis–NIR spectroscopy, water-dispersed functionalized MWNTs revealed the highest absorbance value. Cytotoxicity of MWNTs was investigated before and after functionalization in breast cancer (MDA-MB-231) and human vein endothelial (HUVEC) cells. Relatively low-toxicity results were obtained in functionalized MWNTs and cellular uptake of MWNTs were corrected with fluorescent imaging of cells and cell lysates. Protein-loading capacity of fsMWNTs (functionalized short-length multi-walled carbon nanotubes) was evaluated by using bovine serum albumin (BSA) and with an equal amount of fsMWNTs and BSA; 36% binding yield was obtained. Protein corona after covalent functionalization potentially lowered cytotoxicity up to 6%.