Electrochemical sensors are essential for point-of-care testing (POCT) and wearable sensing devices. Establishing an efficient electron transfer route between redox enzymes and electrodes is key for converting enzyme-catalyzed reactions into electrochemical signals, and for the development of robust, sensitive, and selective biosensors. We demonstrate that the site-specific incorporation of a novel synthetic amino acid (2-amino-3-(4-mercaptophenyl)propanoic acid) into redox enzymes, followed by an S-click reaction to wire the enzyme to the electrode, facilitates electron transfer. The fabricated biosensor demonstrated real-time and selective monitoring of tryptophan (Trp) in blood and sweat samples, with a linear range of 0.02–0.8 mm. Further developments along this route may result in dramatic expansion of portable electrochemical sensors for diverse health-determination molecules.