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Polyunsaturated fatty acid (PUFA) in breast milk provides physiological benefits for offspring and is closely related to endogenous biosynthesis in lactating women. Few studies have addressed the association between fatty acid desaturase (<i>FADS</i>) gene expression patterns and fatty acids in breast milk. This research aimed to explore the differences in PUFA levels among breast milk groups with different levels of <i>FADS</i> gene expression and provide a scientific basis for precision nutrition strategies. A total of 50 healthy women 42–45 days postpartum were included in this study. A basic information questionnaire and breast milk samples were collected. Eight types of PUFA were detected, and RNA was extracted from breast milk. The transcription level of the <i>FADS</i> gene was detected using real-time quantitative PCR. Significant differences in the content of gamma-linolenic acid and eicosatrienoic acid (C20:3n6) were found in breast milk among <i>FADS1</i> gene transcription groups (<i>p</i> = 0.009, <i>p</i> = 0.042, respectively). No significant differences in PUFA were found among the <i>FADS2</i> and <i>FADS3</i> gene expression groups. The results demonstrated that n-6 PUFA was associated with the mRNA expression levels of the <i>FADS1</i> gene. They are of great significance in developing new methods and diets to optimize infant feeding using breast milk.