High-precision analytical methods to detect fumonisin B₁ (FB₁) in a limited range of biological samples have recently been reported. Here an analytical method for FB₁ for use in a wider range of tissue and body fluid samples, such as the plasma, kidney, liver, urine, and bile was developed. Pigs were used in this investigation and administered with FB₁ via osmotic pumps. Samples were extracted with a formic acid/acetonitrile/water mixture, purified on a phospholipid removal solid-phase extraction column, and analyzed by ultra-high performance liquid chromatography-electrospray ionization/tandem mass spectrometry (UHPLC-ESI/MS/MS). The mean recovery of FB₁ in the plasma, kidney, liver, and urine ranged from 80%–115%, and the corresponding relative standard deviation (RSD) was below 10%. The matrix effects resulted in ionization suppression in the bile samples, but these effects were stable and constant, and could be accounted for; the mean recovery and RSD for the bile samples was 10% and <10%, respectively. Using this analytical method, FB₁ was precisely detected in pigs and its levels decreased in the order of urine>bile>kidney=liver>plasma, which implied fast excretion. The detection and quantitation limits (1 and 3 ng g⁻¹, respectively) alongside the other results indicated that this method was suitable for the analysis of FB₁ in various types of biological samples.