This study aims to investigate the effects of galacto-oligosaccharides (GOS) combined with human milk oligosaccharide 2’-fucosyllactose (2’-FL) on the absorption efficiency of amino acids, fatty acids, and minerals in infant formula. An in vitro simulated digestion of infant formula was carried out in a dynamic infant gastroduodenal digestion system. After 0, 30, 60, 120, and 180 min, the transport rates and apparent permeability coefficients (P_app) of nutrients in digestion products were evaluated using the Caco-2 cell model. The results showed that the transport rates of total free amino acids (28.08%–36.98% vs. 24.81%–33.67%), butyric acid (11.02%–16.80% vs. 9.66%–13.29%), caprylic acid (8.22%–14.49% vs. 7.41%–13.03%), capric acid (7.31%–12.66% vs 6.21%–11.53%), calcium (12.02%–23.80% vs. 9.66%–19.29%), and zinc (9.22%–18.49% vs. 7.41%–15.03%) and corresponding P_app were significantly higher in infant formula with added GOS and 2’-FL than in infant formula without added GOS or 2’-FL (P < 0.05), while the differences in the transport rates and P_app of iron and magnesium were not significant (P > 0.05). These data suggest that GOS and 2’-FL can significantly enhance the in vitro absorption efficiency of free amino acids, medium- and short-chain fatty acids, calcium, and zinc in infant formula digests by intestinal cells, independent of regulating the intestinal microbiota.