This work describes the application of a three-dimensional gold nanoelectrode ensembles (GNEE) for monitoring L-dopa in standards and human urine samples using flow injection analysis (FIA) with amperometric detection. Analytical results reveal that the GNEE exhibited better electrocatalytic activity than a gold disk or glassy carbon electrode. Under optimal conditions of L-dopa analysis at GNEE, the calibration plot has a linear range of 5-300 ng/mL with a coefficient of variation (CV) of 3.1% in pH 7.0 phosphate buffer saline (PBS, pH 7.0). The detection limit was 3.0 ng/mL for FIA. The high precision and sensitivity of GNEE provides a feasible means of directly determining l-dopa in urine samples.