L-Cyteine is a form of amino acid found in human body. Retaining the exact quantity of L-Cyteine is important for better functioning of the body. A novel hybrid TiO2 nanostructure (H-TNTs) was prepared on both sides of Ti sheet using the first-step anodization in used (residual) ethylene glycol (EG) based electrolyte. The H-TNTs was explored as an enzyme-free electrochemical biosensor for the detection of L-Cysteine (L-Cyst) and hydrogen peroxide (H2O2). Structural analysis revealed that annealed H-TNTs was anatase with uniform tube morphology and narrow pore size distribution of the top thin nanoporous layer. Electrochemical measurements demonstrated excellent electrocatalytic activities of H-TNTs with sensitivity and rapid response of L-Cyst (0.9914 μA mM−1 cm−2, <2 s) and H2O2 (85.3 μA mM−1 cm−2, < 5 s) respectively. The DFT analysis described that TNT has greater affinity towards L-Cyst and H2O2 with stronger binding distances after the adsorption. The higher negative Eads values suggesting the stable and chemisorptions nature of the H-TNTs. The density of states (DOS) results show that Egap of TNT was significantly reduced after both molecules adsorption. The fabricated electrochemical biosensor exhibited decent stability, excellent reproducibility, and good resistance to interfering molecules showing great potential, as a unique, non-enzymatic electrochemical sensor for future medical applications.