Sterigmatocystin (STC) is a mycotoxin produced by several species of Aspergillus and other fungi. STC is a precursor of the carcinogen aflatoxin B₁ and exerts carcinogenic, teratogenic, and mutagenic effects. A reliable GC-MS-based analytical method using on-column injection was developed and validated to determine STC content in grain. In this method, STC was extracted with acetonitrile (84 %), and the filtered extract was diluted with phosphate buffer before immunoaffinity column (IAC) cleanup. After elution and N₂ evaporation, the sample containing STC was dissolved in acetone and injected into the GC-MS system without derivatization. Separation of STC was carried out by a capillary column (0.25 mm i.d. × 30 m, 0.25 μm) with a deactivated pre-column (0.53 mm i.d. × 0.6 m). The matrix effect was investigated in maize, wheat, and rice, and an insignificant matrix effect was observed after IAC cleanup. The calibration curve was linear in the range of 20–300 pg (equivalent to 8–120 μg/kg in grain) with a coefficient of determination (R ²) of 0.999, and the relative standard deviation of repeatability (RSDr) was less than 10 %. The limit of detection (LOD) of the method was 6 pg (equivalent to 2.4 μg/kg in grain), and the limit of quantification (LOQ) was 20 pg (equivalent to 8 μg/kg). The method described in this report is sensitive, reliable, and can be used to monitor STC contamination in grain.