The performance of an amperometric biosensor constructed by associating tyrosinase (Tyr) enzyme with the advantages of a 3D gold nanoelectrode ensemble (GNEE) is evaluated in a flow-injection analysis (FIA) system for the analysis of Levodopa (L-dopa). GNEEs were fabricated by electroless deposition of the metal within the pores of polycarbonate track-etched membranes. A simple solvent etching procedure based on the solubility of polycarbonate membranes is adopted for the fabrication of the 3D GNEE. Afterwards, enzyme was immobilized onto preformed self-assembled monolayers of cysteamine on the 3D GNEEs (GNEE-Tyr) via cross-linking with glutaraldehyde. The experimental conditions of the FIA system, such as the detection potential (-0.200 V (vs. Ag/AgCl)) and flow rates (1.0 mL min(-1)) were optimized. Analytical responses for L-dopa were obtained in a wide concentration range between 1 x 10(-8) mol L-1 and 1 x 10(-2) mol L-1. The limit of quantification was found to be 1 x 10(-8) mol L-1 with a resultant % RSD of 7.23% (n = 5). The limit of detection was found to be 1 x 10(-9) mol L-1 (S/N = 3). The common interfering compounds namely, glucose (10 mmol L-1), ascorbic acid (10 mmol L-1), and urea (10 mmol L-1) were studied. The recovery of L-dopa (1 x 10(-7) mol L-1) from spiked urine samples was found to be 96%. Therefore, the developed method is adequate to be applied in the clinical analysis.