Mycotoxins are key factor in safeties of food and feed. However, most previous studies about dairy ruminants have focused on the biochemical parameters or milk production. Little is known about the milk micro-profiles, such as fatty acids and amino acids, under mycotoxin exposure. With taking advantage of the well-established chromatography and NMR (nuclear magnetic resonance) analyses, milk micro-profiles and metabolic changes of dairy cow were measured after mycotoxins exposure. The results showed that milk composition of dairy cows are unchanged, but milk aflatoxin M₁ concentration changed along with the mycotoxins addition or clearance. Dairy cows fed cottonseed contaminated with mycotoxins had a significant reduction in unsaturated long-chain FAs in milk, but there was no reduction in the content of short- and medium-chain fatty acids. Interestingly, contaminated cottonseed did not reduce the content of amino acids in milk. Twelve metabolites were showed significant difference among the 3 groups. These metabolites are mainly involved in the metabolic pathways of phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism and valine, leucine and isoleucine biosynthesis. This may indicate that amino acid metabolism is affected by mycotoxins intakes. In conclusion, these results suggest that it is important not only to study milk composition, but also to pay attention to micro-indicators (FAs and amino acids) in the study of the harm of mycotoxins to dairy cows.