We have prepared a pyrolytic graphite electrode (PGE) whose surface is covered with a thin film of a nano-mixture of graphite/diamond (NGD). The electrode is shown to be capable of electrochemically sensing of tryptophan (TRP) and 5-hydroxytryptophan (HTRP). The presence of the NGD film resulted in a remarkable increase in the peak currents and sharpness of the waves so that submicromolar concentrations of TRP and HTRP become detectable. Potential scan rates, the pH of the solution, the accumulation conditions and the amount of the modifier were optimized via cyclic voltammetry. Linear sweep voltammetry, under optimized accumulation time and in open circuit operation, was applied to the determination of TRP and HTRP with detection limits (S/N = 3) of 30 nM (TRP) and 6 nM (HTRP). The electrode can be easily prepared, displays high sensitivity, sharp peaks, long-term stability, and remarkable voltammetric reproducibility and repeatability. These properties make the sensor suitable for the trace analysis of TRP and HTRP in pharmaceutical and clinical preparations.