A simple methodology of synthesizing sub-micrometer sized polyaniline (PANI) spheres using gold electrodes with thiourea linked glycolipid self-assembly for the selective detection of dopamine (DA) is reported here. The synthesis was carried out using a potentiodynamic polymerization method. The self-assembled thiourea linked glycolipid on the gold electrodes and the same lipid acting as the surface directing agent for the formation of polyaniline microspheres is the highlighting factor in this work. The biocompatible sub-micrometer polyaniline spheres are characterized using scanning electron microscopy and transmission electron microscopy studies. Amperometry and electrochemical impedance spectroscopy were employed to estimate the concentration of dopamine. The amperometric studies reveal a linear range of ~ 1 to 640 μM, sensitivity of 370 μA cm⁻² μM⁻¹, a very low detection limit of 10 nM, and a response time of ~ 5 s. The interference from L-Dopa, ascorbic acid and uric acid has been minimized on account of the Nafion (Nf) coating and selectivity of the electrode. Real sample analysis was carried out using standard addition method.