Sensitive detection of Staphylococcus aureus enterotoxin B (SEB) is of importance for preventing food poisoning from threatening human health. In this work, an electrochemical and colorimetric dual-signal detection assay of SEB was developed. The probe (Ab2/AuPt@Fe-N-C) was bound to SEB captured by Ab1, where the Ab2/AuPt@Fe-N-C triggered methylene blue degradation and resulted in the decrease of electrochemical signal. Furthermore, the probe catalyzed the oxidation of 3, 3’, 5, 5’-tetramethyl biphenyl to generate a colorimetric absorbance at 652 nm. Once the target was captured and formed a sandwich-like complex, the color changed from colorless to blue. SEB detection by colorimetric and electrochemical methods showed a linear relationship in the concentration ranges of 0.0002–10.0000 and 0.0005–10.0000 ng/mL, with limits of detection of 0.0667 and 0.1670 pg/mL, respectively. The dual-signal biosensor was successfully used to detect SEB in milk and water samples, which has great potential in toxin detection in food and the environment.