Levodopa, one of the prescribed medications in Parkinson disease, is electrochemically determined here. A Cu/TiO2 nanocomposite/room temperature ionic liquid/2-(ferrocenylethynyl)fluoren-9-one modified carbon paste electrode (Cu/TiO2-IL- 2FF/CPE) was used for the electrochemical determination of levodopa. The electro-oxidation mechanism of levodopa at Cu/TiO2-IL-2FF/CPE was also studied. The obtained data showed that the electro-oxidation of levodopa at the Cu/TiO2-IL-2FF/CPE is greatly facilitated, which is attributed to high electrical conductivity of Cu/TiO2 nanocomposite, room temperature ionic liquid and 2-(ferrocenylethynyl)fluoren-9-one. The Cu/TiO2-IL-2FF/CPE showed a good electron mediating behavior followed by well separated oxidation signals of levodopa and tyrosine. Differential pulse voltammetric peak current showed a linear relationship corresponds to the concentrations of levodopa in a linear range of 0.03 to 700.0 μM with detection limit of 12.0 nM. The Cu/TiO2-IL-2FF/CPE shows excellent ability to determination of levodopa and tyrosine in real samples.