We present a surface-enhanced Raman probe (SERS) platform for the determination of a prohibited substance, recombinant erythropoietin (rEPO), in urine matrix, using nanoparticles as substrate. Rod-shaped gold nanoparticles (GNR) were modified with a Raman label and an antibody as SERS probe. We developed two SERS-based immunoassays for detection and quantification of rEPO in urine. In the first assay, rEPO was determined by a sandwich assay with gold surfaces and GNR. In the second assay, rEPO was extracted by using core shell-structured magnetic iron oxide gold nanoparticles, and again sandwich assay was performed by using GNR. We also demonstrated the ability of the proposed method to discriminate rEPO and urinary erythropoietin (uEPO). A good linear correlation was obtained between logarithms of rEPO concentrations in urine and Raman intensities within the range of 10(-1)-10(3) pg mL(-1) rEPO concentrations. Detection limits which are smaller than 0.1 pg mL(-1) levels were achieved owing to the high extractive performance of the nanoextraction techniques.