Avoiding interference and realizing the precise detection of mycotoxins in complex food samples is still an urgent problem for surface-enhanced Raman spectroscopy (SERS) analysis technology. Herein, a highly sensitive and specific aptasensor was developed for the anti-interference detection of Ochratoxin A (OTA). In this aptasensor, 4-[(Trimethylsilyl) ethynyl] aniline was employed as an anti-interference Raman reporter to prove a sharp Raman peak (1998 cm−1) in silent region, which could avoid the interference of food bio-molecules in 600–1800 cm−1. 4-TEAE and OTA-aptamer were assembled on Au NPs to serve as anti-interference SERS probes. Meanwhile, Fe3O4 NPs, linked with complementary aptamer (cApts), were applied as capture probes. The specific binding of OTA to aptamer hindered the complementary binding of aptamer and cApt, which inhibited the binding of SERS probes and capture probes. Hence, the Raman responses at 1998 cm−1 were negatively correlated with the OTA level. Under the optimum condition, the aptasensor presented a linear response for OTA detection in the range of 0.1–40 nM, with low detection limits of 30 pM. In addition, the aptasensor was successfully applied to quantify OTA in soybean, grape and milk samples. Accordingly, this anti-interference aptasensor could perform specific, sensitive and precise detection of OTA in real samples, and proved a reliable reference strategy for other small-molecules detection in food samples.