L-DOPA, or l-3,4-dihydroxyphenylalanine is an aromatic amino acid, which plays a significant role in human metabolism as a precursor of important neurotransmitters. We develop a fast and simple colorimetric method for the detection of L-DOPA in biological fluids. The method is based on the reduction of silver ions with L-DOPA and the subsequent formation of L-DOPA stabilized silver nanoparticles (Ag NPs). In this novel approach, L-DOPA works as both reducing and stabilizing agent, which provides selectivity and simplifies the procedure. HR-TEM images show very narrow Ag NPs distribution with an average size of 24 nm. Such sensor design is suggested for the first time. We also calculate vertical ionization potential, vertical electron affinity, and Gibbs free energy change of different ionic forms of L-DOPA and amino acids at the M06-2X/def2-TZVP level for the gas phase in comparison with that of silver. A model of silver ions reduction by aromatic amino acids is proposed: the ionic forms with charge −1 are suggested to reduce silver ions. High selectivity against aromatic amino acids, dopamine and serotonin is achieved by tuning pH and involving two L-DOPA forms with charged both hydroxyphenolate and carboxylate groups in the stabilization of uniform-sized Ag NPs. The method is applicable for the determination of L-DOPA in human serum with the 50 nM limit of detection and the linear range up to 5 μM. Ag NPs formation and coloring the solution proceeds in a few minutes. The suggested colorimetric method has potential application in clinical trials.