Gold-grafted melamine sponge (AuSp) was fabricated and applied as surface-enhanced Raman spectroscopy (SERS) substrate for enzyme-linked immunoassay of 2 typical mycotoxins, zearalenone (ZEN) and T-2 toxin. The gold nanoparticles were in situ grown on the sponge, and the product showed good effect in enhancing Raman signal and good stability. COMSOL and Gaussian simulations were used to analyze the enhancing effect. Some of the experimental conditions that may affect the determination results were studied in order to meet the maximum response to mycotoxins. At optimized conditions, the limits of detection of the method were 1 μg·kg and 0.05 μg·kg for ZEN and T-2 toxin, with linear ranges of 5-100 μg·kg and 0.1-20 μg·kg, respectively, and relative standard deviations lower than 7.0%. The established method was used to determine ZEN and T-2 toxin in six kinds of cereal samples, the recoveries were 85-120%, and relative errors were less than 13.0%. These results indicated the accuracy of the established method. After 1-year storage, signal strength of AuSp for mycotoxins determination decreased by less than 10%, showing stability over a long time. Our work provided a new method for SERS determination of mycotoxins, which reduces the complexity of substrate preparation and the sample consumption compared with traditional methods, and thus has potential for on-line SERS determination.