A novel modified carbon paste electrode based on TiO₂ hollow spheres (TOHS), multi-walled carbon nanotubes (MWCNT) and poly-aspartic acid (P-ASP) films (P-ASP/TOHS/MWCNT/CPE) was developed and used as a selective electrochemical sensor for simultaneous determination of levodopa (LD) and acetaminophen (ACT) in the presence of ascorbic acid (AA). The measurements were carried out by the application of differential pulse voltammetry (DPV), cyclic voltammetry (CV) and chronoamperometry (CA) methods. The prepared electrode presented rapid acceleration in electron transfer reactions of LD and ACT and showed no significant interference of AA as electro-active coexistent compounds with LD and ACT in biological systems. The DPV data showed that under optimal conditions, the obtained anodic peak currents were linearly dependent on the LD and ACT concentrations in the range of 0.4–450 and 0.1–300 μM, respectively. The fabricated sensor offered numerous advantages such as convenient preparation, good stability and high sensitivity towards LD and ACT determination. The applicability of the modified electrode was also demonstrated for simultaneous determination of LD and ACT in human urine.