Discovering alternative analytical techniques is crucial for practical applications; thus, this work aims to develop an innovative and simple electrochemical sensor for melanoma and the clinical diagnosis of related disorders by the simultaneous determination of 3,4-dihydroxy-L-phenylalanine (L-DOPA) and L-tyrosine (L-Tyr). The fabrication is based on the layer-by-layer electrodeposition of poly L-proline (poly(L-pro)) and nanodiamond (ND) onto a screen-printed graphene electrode (SPGE). The poly(L-pro)/ND/SPGEs were morphologically characterized by scanning electron microscopy, energy-dispersive X-ray spectrometry, and Raman spectroscopy followed by electrochemical investigation using cyclic voltammetry, differential pulse voltammetry, chronoamperometry, and electrochemical impedance spectroscopy. These modifier-based electrodes pave a feasible way to unlock the coexisting interfering substances from screen-printing ink composition and improve the sensitivity. Additionally, computational chemistry calculations were performed to fully comprehend the sensing behavior on both target analytes. Under optimal conditions, the developed sensor provided linear concentration ranges of 0.075-50 μM, with a detection limit of 0.021 μM for L-DOPA, and 2.5-120 μM with a detection limit of 0.74 μM for L-Tyr. To demonstrate the reliability of the poly(L-pro)/ND/SPGE in practical application, it was successfully applied to the determination of these analytes in human urine and blood serum samples, with satisfactory recovery ranges (81.73-110.62% for L-DOPA and 82.17-110.01% for L-Tyr) and relative standard deviations (0.69-9.90% for L-DOPA and 0.40-9.55% for L-Tyr). Due to its simplicity, long-term stability (> 87.8% of their initial currents after 35 days), and portability, the developed sensor is a promising alternative analytical method for on-site clinical monitoring.© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.