Poly(3,4-ethylenedioxythiophene) (PEDOT) modified with ferrocene carboxylic acid (FC1), ferrocene (FC2), and cobaltocene (CC) is introduced as a sensor electrode. FC or CC was “sandwiched” between two layers of PEDOT in the presence of sodium dodecyl sulfate (PEDOT/mediator/PEDOT…SDS). The composite electrodes were evaluated for the electrocatalytic oxidation of dopamine (DA). The FC1 mediator showed the highest rate for electron transfer and enhanced electrocatalytic activity. This is due to the inclusion of the ferrocenium ion and the polar substituted –COOH group in the matrix which increases the electronic conduction of the film. SDS enhanced the preconcentration/accumulation of DA ions at the surface that resulted in enhanced detection. Detection limit in human urine was 0.069 μmol L−1 in the linear dynamic range of 6–300 μmol L−1, with satisfying recovery results. The PEDOT/FC1/PEDOT…SDS composite was used for simultaneous determination of dopamine in the presence of ascorbic acid (AA) and uric acid (UA).