Intestinal permeability is a key factor affecting the bioavailability and physiological efficacy of docosahexaenoic acid (DHA) encapsulated in microcapsules. However, how the DHA microcapsules are transformed and the components absorbed across the small intestinal membrane has seldom been examined previously. In this study, an ex vivo absorption model based on the permeability of the rat small intestine was established to evaluate the intestinal absorption of DHA microcapsules with five formulations after gastrointestinal digestion in vitro. For pure glucose solutions, the apparent permeability coefficient (Papp) increased from 5.70 ± 0.60 × 10-6 cm s-1 at 5 mg mL-1 to 20.25 ± 0.88 × 10-6 cm s-1 at 30 mg mL-1 and decreased to 15.73 ± 0.91 × 10-6 cm s-1 at 100 mg mL-1. The Papp values obtained using the ex vivo model are comparable to those reported in the human jejunum. For algal oil DHA microcapsules with whey protein as the wall material (A-WP-DHA) after in vitro digestion, the Papp of glucose released was 3.81 × 10-6 cm s-1 with an absorption ratio of 59.55% in the ex vivo model, significantly lower than that from the in vitro porcine casing model. The Papp and absorption ratio varied little among the in vitro dialysis models with different molecular weight cut-off values. A similar trend was observed for the absorption of amino acids. However, the absorption ratio (26.6%) was the highest in the ex vivo model for free fatty acids (FFAs) released from the microcapsules due to the rapid accumulation of compounds on the inner wall of the intestinal sac. In addition, the DHA microcapsules with algal oil as the DHA source (36.40%) exhibited a higher absorption ratio of FFAs than that from tuna oil (14.26%) in the ex vivo model. The wall material compositions seemed to have little effect on FFA absorption. The present study is practically meaningful for the future formulation of DHA microcapsules with enhanced absorption.