Pterins are heterocyclic compounds which play an essential role in physiology and serve as sensitive biomarkers of pathological processes, including immune disorders, inflammation, and cancer. However, their detection in complex biological matrices remains challenging due to low physiological concentrations and strong influence of other biomolecules. In this study, we demonstrated the application of surface-enhanced Raman scattering (SERS) with aggregated gold nanoparticles for the sensitive and selective detection of pterins, particularly neopterin, in human serum. The proposed approach based on strong enhancement of characteristic Raman bands at 692 and 1713 cm−1, allowed pterins differentiation from amino acids and other serum components. Quantitative analysis of neopterin was achieved using intensity ratios (I694/I1663 and I1712/I1663) with the Amide I band as an internal reference, resulting in the linear calibration curves (R2 > 0.99) in the range of 30–240 nM. The detection limit was estimated at 16 nM. Compared to conventional chromatographic and mass spectrometric methods, SERS offers the advantages of minimal sample preparation, rapid analysis, and the possibility of in situ diagnostics. These findings highlight the potential of SERS as a promising analytical tool for clinical diagnostics and biomedical research involving pterin biomarkers.