An innovative approach based on photothermal contrast to enhance analytical sensitivity of enzymatic phenylalanine detection at a solid surface was demonstrated. Gold nanoparticles in situ produced by the reduced form of nicotinamide adenine dinucleotide, which is generated by an enzymatic reaction, exhibit an excellent photothermal effect upon green-light stimuli. The enzymatic reaction and the nanoparticles process formation were optically investigated. A sensing range of 10–1000 μM with a detection limit of 3.6 μM for phenylalanine detection were demonstrated. The testing with blood samples collected from phenylketonuria patients together with concurrent comparison through tandem mass spectrometry have confirmed the good analytical performances and the robustness and utility of the proposed photothermal-contrast approach for further integration on clinical devices.