This paper deals with adsorption behavior of ruthenium on Tri-n-butyl phosphate functionalized Multi Walled Carbon Nanotubes (MWCNTs-COO-TBP) from aqueous solution. Adsorption method is significant for trace levels (expected level) removal of ruthenium in bioassay (urine) samples which is mandatory requirement of radiation workers in nuclear environment. Multi Walled Carbon Nanotubes (MWCNTs) were initially converted to carboxylated Multi Walled Carbon nanotubes (MWCNTs-COOH) and subsequently treated with Tri-n-butyl phosphate to MWCNTs-COO-TBP. The synthesized MWCNTs-COOH and MWCNTs-COO-TBP were confirmed by Fourier Transform Infrared Spectroscopy (FTIR). Spectroscopic and microscopic techniques such as X-Ray Diffraction (XRD), Raman Spectroscopy, Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy (SEM-EDS) and High Resolution Transmission Electron Microscopy (HR-TEM) were used for its characterization before and after adsorption. Various adsorption parameters such as contact time, pH, metal ion concentration and temperature were studied. Langmuir adsorption isotherm model and Pseudo second order kinetics model were found to be best fitted in terms of standard deviation and regression coefficient. Thermodynamics parameters such as enthalpy (ΔH°), entropy (ΔS°), Gibbs energy (ΔG°), activation energy (Ea) and sticking probability (SP*) were calculated. The adsorption capacity was calculated and found to be 141.23 mg.g−1.Moreover, quantification of defects, strains with charge doping states were calculated from Raman mapping and correlation plot. Display omitted •Synthesis and characterization of Tri-n-Butyl Phosphate functionalized Multi Walled Carbon Nanotubes (MWCNTs-COO-TBP).•Detailed study on adsorption of ruthenium on MWCNTs-COO-TBP and its kinetics, thermodynamics and isotherm.•Functionalization of MWCNTs by TBP and its adsorption for ruthenium, confirmed by FTIR and EDS analysis.•Quantification of defects and strains with charge doping states were calculated from Raman mapping and correlation plot.•The potential application of MWCNTs-COO-TBP for adsorption of Ru from aqueous solution was explored.