A chemically modified glassy carbon (GC) electrode was developed as an amperometric sensor for detection of biological thiols. The electrode was modified by inclusion of co‐enzyme pyrroloquinoline quinone (PQQ) and a co‐catalyst of oxidized single wall carbon nanotubes (Ox‐SWNT) into a gold polypyrrole (Au‐PPy) nanocomposite matrix. The electrode (PQQ/Ox‐SWNT/Au‐PPy/GC) was characterized using scanning electron microscopy and cyclic voltammetry. Optimal conditions for the PQQ/Ox‐SWNT/Au‐PPy/GC electrode were determined and then utilized for the amperometric detection of L‐cysteine, N‐acetyl‐L‐cysteine, L‐penicillamine and D, L‐glutathione. The electrochemical response for each thiol in pH 3.2 citrate phosphate buffer at +450 mV (vs. Ag/AgCl) was found to be linear with limit of detections (LOD, S/N=3) ranging from 0.50 µM for L‐penicillamine to 1.55 µM for D, L‐glutathione with sensitivities of 30.2 nA/µM and 3.6 nA/µM respectively. The electrode design is simple and easy to construct using a minimum amount of co‐enzyme and co‐catalyst, resulting in detection methods with very good stability and improved sensitivity for thiol detection.