Nervonic acid (24:1 n-9, NA) plays a crucial role in the development of white matter, and it occurs naturally in human milk. This study aims to quantify NA in human milk at different lactation stages and compare it with the NA measured in infant formulae. With this information, optimal nutritional interventions for infants, especially newborns, can be determined. In this study, an absolute detection method that uses experimentally derived standard curves and methyl tricosanoate as the internal standard was developed to quantitively analyze NA concentration. The method was applied to the analysis of 224 human milk samples, which were collected over a period of 3–30 days postpartum from eight healthy Chinese mothers. The results show that the NA concentration was highest in colostrum (0.76 ± 0.23 mg/g fat) and significantly decreased (p < 0.001) in mature milk (0.20 ± 0.03 mg/g fat). During the first 10 days of lactation, the change in NA concentration was the most pronounced, decreasing by about 65%. Next, the NA contents in 181 commercial infant formulae from the Chinese market were compared. The NA content in most formulae was <16% of that found in colostrum and less than that found in mature human milk (p < 0.05). No significant difference (p > 0.05) was observed among NA content in formulae with different fat sources. Special attention was given to the variety of n-9 fatty acids in human milk during lactation, and the results indicated that interindividual variation in NA content may be primarily due to endogenous factors, with less influence from the maternal diet.