The kinetics of aflatoxin B (AFB) and its carry-over as aflatoxin M (AFM) in milk as well as the toxin loads in the tissue of dairy cows were assessed through a repetitive feeding trial of an AFB-contaminated diet of 4 μg kg body weight (b.w.) for 13 days. This was followed by a clearance period that ended with a single dose trial of an AFB-contaminated diet of 40 μg kg b.w. An ultra-high performance liquid chromatography tandem mass spectrometry method was developed and successfully validated by the determination of linearity ( ≥ 0.990), sensitivity (lower limit of quantification, 0.1-0.2 ng ml), recovery (79.5-111.2%), and precision relative standard deviation (RSD) ≤14.7%) in plasma, milk, and various tissues. The repetitive ingestion of AFB indicated that the biotransformation of AFB to AFM occurred within 48 h, and the clearance period of AFM in milk was not more than 2 days. The carry-over rate of AFM in milk during the continuous ingestion experiment was in the range of 1.15-2.30% at a steady state. The kinetic results indicated that AFB reached a maximum concentration of 3.8 ± 0.9 ng ml within 35.0 ± 10.2 min and was slowly eliminated from the plasma, with a half-life time (T) of 931.1 ± 30.8 min. Meanwhile, AFM reached a plateau in plasma (0.5 ± 0.1 ng ml) at 4 h after the ingestion. AFB was found in the heart, spleen, lungs, and kidneys at concentrations of 1.6 ± 0.3, 4.1 ± 1.2, 3.3 ± 0.9 and 5.6 ± 1.4 μg kg, respectively. AFM was observed in the spleen and kidneys at concentrations of only 0.7 ± 0.2 and 0.8 ± 0.1 μg kg, respectively. In conclusion, the kinetics and biotransformation of AFB in dairy cows were determined using the developed UHPLC-MS/MS method, and the present findings could be helpful in assessing the health risks to consumers.