The production and distribution of 2,5-dimethylpyrazine (2,5-DMP), a key volatile flavor compound, are associated with the frying of skipjack tuna steaks. In this study, an intestinal villus-like chip was fabricated to quantitatively detect 2,5-DMP in multiphase systems based on surface-enhanced Raman spectroscopy (SERS). The chip exhibited excellent SERS performance with an enhancement factor of 1.16 × 108, excellent uniformity and reproducibility, and low detection limits of 6.49 pg/mL, 0.182, and 0.920 ng/mL for 2,5-DMP solid, liquid, and gas models, respectively. The results indicated the 2,5-DMP content with growth rates in the order of frying steam > frying steaks > frying oil, and the 2,5-DMP content in frying steam was linearly correlated (R2 = 0.992) with the trend of the acid value and total polar compounds in frying oil. Therefore, this strategy for achieving the reliable detection of 2,5-DMP can assist in monitoring the frying process and food safety.