Phenylalanine has long been recognized as a reliable biomarker for the diagnosis and monitoring of phenylketonuria (PKU), a rare genetic disease characterized by the impairment of phenylalanine hydroxylase. As part of the current efforts toward the design of point-of-care devices for the day-to-day management of PKU, we set up a coupled enzymo–plasmonic assay of phenylalanine based on the combination of the nicotinamide adenine dinucleotide (NAD+)-dependent enzyme phenylalanine dehydrogenase and gold nanoparticles to visually detect physiologically meaningful concentrations of phenylalanine and quantify them with a lightweight portable spectrophotometer compatible with smartphone readout. The principle of the assay relies on the dissolution of gold nanoparticles (AuNPs) induced by mixtures of HAuCl4 and cetyltrimethylammonium bromide (CTAB) and its inhibition by (enzymatically produced) NADH, establishing a relationship between optical readout and phenylalanine concentration with a limit of detection of ca. 1 μM. The transposition of this assay to the analysis of serum samples containing phenylalanine concentrations spanning from 60 to 2000 μmol/L was achieved, validating the nanobiosensor as a promising tool for PKU management.