Abstract An enzymatic-colorimetric method has been developed based on the reaction between l-phenylalanine (l-Phe) and the l-amino acid oxidase (LAAO) in the presence of Au(III), which has led to the formation of gold nanoparticles. The intensity of the localized surface plasmon resonance (LSPR) band of the generated nanoparticles (550 nm) can be related to the concentration of l-Phe in the sample. The mechanism of the LAAO-l-Phe enzyme reaction in the presence of Au(III) has been studied through the evaluation and optimization of experimental conditions. These studies have reinforced the hypothesis that the catalytic center of the enzyme helps the Au(III) reduction and, thanks to the protein, the Au0 form is stabilized as gold nanoparticles (AuNPs). In the calibration study, a sigmoidal relationship between the concentration of the substrate and the LSPR of the nanoparticles was observed. The linearization of the signal has allowed the determination of l-Phe in the range from 17 to 500 µM with an RSD% (150 μM) of 4.8% (n = 3). The method is free of other amino acid interference normally found in blood plasma. These highly competitive results open the possibility of further development of a rapid method for l-Phe determination based on colorimetry. Graphical abstract