In order to analyze sterigmatocystin (STC) by surface-enhanced Raman scattering (SERS) spectroscopy, gold substrates were prepared containing nanoparticles dispersed in suspension and immobilized on solid supports. The SERS efficiency of metal substrates was tested with rhodamine 6G (R6G). In colloidal gold suspensions, an intense enhancement of R6G Raman scattering was observed at excitation of 785 nm, while the spectra were overshadowed by rhodamine dye fluorescence at excitation of 532 nm. The presence of an aggregating salt in the colloid did not affect the sensitivity of the R6G measurements. In the spectra of STC in the gold colloid, regardless of the excitation wavelength, STC bands were not observed, but bands of methanol, in which the STC solution was prepared, and bands of citrate ions and their oxidation/degradation products, which remained on the gold nanoparticles surface after the synthesis. Characteristic STC bands of very low intensity were observed for the highest measured STC concentrations in the spectra of colloidal samples aggregated with ammonium sulfate. The spectra of the solid gold substrates on a glass support, taken at 785 nm excitation, were dominated by photoluminescence of glass. Weak Raman scattering of R6G on the gold substrate on glass was observed after excitation at 532 nm. The enhancement of the Raman scattering on the gold nanoparticles immobilized on an aluminum support was not obtained for either R6G or STC.