ZnO nanoflowers (ZnONFs) were synthesized by a simple hydrothermal method which is a convenient, environment-friendly, inexpensive process. The ZnONF suspension was sprayed onto the surface of ITO-coated glass (ZnONFs/ITO), which acts as the working electrode for the determination of levodopa by cyclic voltammetry and differential pulse voltammetry. The effect of reaction regents on the sizes of ZnONFs was investigated. The morphologies and structure of ZnONFs were characterized by scanning electron microscopy and X-ray diffraction analysis, respectively. The results show that the optimized length and diameter of ZnONFs are ~2.5 μm and 50 nm, respectively. The sensitivity and measured limit of detection of the ZnONFs/ITO electrode for levodopa are 0.10 μA/μM and 2.5 μM in the range of 2.5–40 μM, respectively. The electrode can accurately detect levodopa in the presence of uric acid. Meanwhile, the electrode shows good repeatability and stability.