Parkinson's disease is the most common neurodegenerative disorders in human mainly due to insufficient norepinephrine supply. Hence, determination of trace level of norepinephrine (NE) in presence of interfering compounds in biological fluids such as paracetamol (APAP), tyrosine (TR), and ascorbic acid (AA) is essential to diagnosis some psychiatric and neurological disorders diseases. A layer-by-layer stacked composite sensor based on crown ether (crown), Nafion (R) (NF), poly (3-4-ethylene dioxythiophene) (PEDOT) and multi-walled carbon nanotubes (CNT) was fabricated (GC/CNT/PEDOT/NF/Crown). Each component possesses a unique feature that contributes to the remarkable electro-catalytic activity of the sensor. Crown ether allows excellent host-guest recognition and binding ability; NF has good ionic conductivity; PEDOT has high electrical conductivity and stability; and CNT has large surface area and mechanical strength. The proposed sensor was successfully applied for electro-oxidation and separation of quaternary mixture of AA, NE, APAP, and TR into four well-defined peaks with separations in potential (Delta E-p) of: 210, 163 and 257 mV for AA-NE, NE-APAP and APAP-TR, respectively. The separation of these compounds in real human blood serum samples was also successful. Detection limits were 8.46 nmol L-1, 0.0862 nmol L-1, 0.0627 nmol L-1 and 0.429 nmol L-1 in linear dynamic ranges of 0.1-30 mu mol L-1, 0.007-10 mu mol L-1, 0.005-8 mu mol L-1 and 0.06-20 mu mol L-1, for AA, NE, APAP and TR, respectively.