Efficient and low-cost surface-enhanced Raman scattering (SERS) substrates based on Au coated zinc oxide layers for the detection of neopterin were prepared. These substrates showed high sensitivity to p-mercaptobenzoic acid (p-MBA) at a low concentration of 10(-9) M and an enhancement factor of over 10(7) was achieved. The uniform density of SERS-active "hot-spots" on a Si/ZnO/Au surface results in high reproducibility towards detecting p-MBA at 50 different, randomly selected positions on a single substrate (RSD = 9%) and on six different SERS substrates prepared under identical conditions (RSD = 11%). These SERS substrates show good performance in the detection of neopterin, a biologically important molecule whose concentration levels reflect the stage of activation of the cellular immune system, which is of value in the studies of pathogenesis and progression of various diseases. The detection limit is found to be as low as 1.4 nmol L(-1) in blood plasma, which is comparable to that of classic ELISA methods. The average relative standard deviation (RSD) of the proposed method is less than 10%. Moreover, this label-free strategy of detection gives exact results over a large range, reflecting clinically relevant neopterin concentrations in body fluids. The detection and quantification of neopterin levels in blood or urine might be useful in clinical practice for monitoring the disease activity during treatment and for early detection of many infections and autoimmune, inflammatory, and malignant diseases.