Fumonisins are mycotoxins from a few species of fungi in the genus Fusarium causing a variety of diseases of both animals and humans. These fungi are commonly found in corn through systematic transfer from their roots. To detect fumonisin occurrence in corn or corn-based food, proper and decent analytical methods are required. In this study, we developed an analytical method for the determination of three types of fumonisins (FB1, FB2, and FB3) in corn flour based on isotope dilution-liquid chromatography tandem mass spectrometric (ID-LC/MS/MS) method. Fumonisins were extracted from corn flour and the extracts were purified with immunoaffinity column before introduced to LC/MS/MS instrument. The experimental parameters, such as kind of extraction solvent, clean-up column, and solvent-to-sample ratio were studied. Solubility and extraction efficiency was better when phosphate buffered saline (PBS) was used as extractant compared to acetonitrile-water or methanol-water solutions. A post-column infusion system was used to investigate the clean-up efficiency of immunoaffinity columns. The amount of fumonisins bound to corn flour matrix could be extracted with PBS. Thus, hydrolysis was not necessary for fumonisin analysis in this study. Selected reaction monitoring (SRM) was employed in the mass spectrometer at m/z 706.5 ⟶ 334.4 (FB1) and m/z 706.4 ⟶ 336.3 (FB2 and FB3). Through the gradient elution of 5 mM ammonium formate in water containing 0.1% formic acid and 5 mM ammonium formate in methanol containing 0.1% formic acid on C18 column, FB1, FB2, and FB3 were successfully separated. The proposed method provided a detection limit range from 0.4 to 0.5 μg/kg and a limit of quantification range from 1.2 to 1.9 μg/kg. Following validation of the developed method, fumonisin level was investigated with additional corn-based products.