Gene expression and post-transcriptional regulation are key mechanisms affecting lactation performance in dairy animals. However, the difficulty of obtaining mammary gland tissue samples from lactating animals has significantly impeded lactation research. Milk fat globules may be a non-invasive way to obtain mammary transcripts. Here, we aimed to reveal the universal rule of the transcript profiles of the milk fat globules and mammary glands from buffaloes and goats by RNA-sequencing analysis. Results showed that, in buffalo, 97 % of mRNAs were expressed in both milk fat globules and mammary glands, with 45 % showing differential expression. Among 6086 lncRNAs and 7010 miRNAs identified, 35 % and 50 % were differentially expressed, respectively. Of 11,631 circRNAs, only 618 showed significant differences. In goat, more than 99 % of mRNAs and 87 % of ncRNAs (including lncRNAs, circRNAs, and miRNAs) were expressed in both milk fat globules and mammary glands, and over 91 % of mRNAs, 96 % of lncRNAs, 98 % of circRNAs, and 86 % of miRNAs showed no significant differences, suggests that the transcripts in milk fat globules exactly reflect that in the mammary gland. This study suggests that milk fat globules are an effective candidate for non-invasive acquisition of mammary gland transcripts, but their applicability needs further study.