Here, Nanocomposites of thallium oxide doped multi-walled carbon nanotube (Tl2O.MWCNT NCs) were prepared by utilizing the wet-chemical method (WCM) in an alkaline phase at low temperature. Different optical procedures (FTIR: Fourier Transform Infra-Red Spectroscopy, XRD: Powder X-ray diffraction, FESEM: Field-Emission Scanning Electron Microscopy, XEDS: X-ray Electron Dispersive Spectroscopy, TEM: Tunneling Electron Microscopy, and XPS: X-ray photoelectron spectroscopy) were used to fully characterize (Optical, structural, crystalline, morphological, and elemental etc.) of the prepared Tl2O.MWCNT NCs. Modification of the thin-layer with NCs onto glassy carbon electrode (GCE) is prepared and applied for the enzyme-free detection of selective and sensitive L-cysteine by electrochemical approach. Using a reliable current-voltage approach, analytical sensing indexes such as sensitivity, LDR, LOD, LOQ, durability, and interference were assessed by fabricated sensor probe (GCE/Tl2O.MWCNT NCs/CPM) in selective detection of L-cysteine in a room condition, whereas nafion was used as conducting polymer matrix (CPM) during the fabrication of GCE with NCs. L-cysteine calibration plot was found to be linear over an extensive range of concentration. The calibration curve was used to calculate the sensing parameters such as sensitivity (316.46 pAμM-1cm-2), LOD down to (~18.90 ± 1.89 pM), and LOQ (63.0 pM) of the prepared sensor. The use of a simple WCM to validate the Tl2O.MWCNT NCs is a good approach for developing a NCs-based sensor for enzyme-free biomolecule identification and detection in the biomedical and health care fields in a broad scale. This proposed sensor (GCE/Tl2O.MWCNT NCs/CPM) is used to detect selective L-cysteine in real biological samples such as human, mouse, and rabbit serum and found acceptable and satisfactory results.