Fatty acid (FA) is the major energy resource in breast milk, which is important for infant development. FAs profiles with sn-2 positional preference were an important part of triacylglycerols due to their better availability. This profile is still not replicated in artificial formulas. This study quantified the FAs profile of total and sn-2 position in human breast milk samples from 103 healthy volunteers during colostrum, transitional, and mature stages. Multicomponent analysis showed significant differences in FAs profiles of different lactation periods, due to that with relative percentage less than 1%. Linoleic acid (LA), mostly located at the sn-1,3 positions of TAGs, was more common in the milk of Chinese women than in western women. The majority of the breast milk did not meet the standard for the ratio of LA/α-linolenic acid for infant formula. FAs related to brain development, mainly at sn-2 in TAGs, were enriched in colostrum. Capric and lauric acids were enriched in transitional and mature breast milk, and capric acid showed sn-1,3 selectivity in TAGs. This study will aid the development of infant formula containing TAGs more similar to human breast milk.