Chrysin (5,7-dihydroxyflavone) is a remarkable flavonoid exhibiting many health-promoting activities, such as antioxidant, anti-inflammatory, and anti-Alzheimer’s disease (AD). Nevertheless, chrysin has been addressed regarding its limited applications, due to low bioaccessibility. Therefore, to improve chrysin bioaccessibility, a colloidal delivery system involving nanoemulsion was developed as chrysin nanoemulsion (chrysin-NE) using an oil-in-water system. Our results show that chrysin can be loaded by approximately 174.21 µg/g nanoemulsion (100.29 ± 0.53% w/w) when medium chain triglyceride (MCT) oil was used as an oil phase. The nanocolloidal size, polydispersity index, and surface charge of chrysin-NE were approximately 161 nm, 0.21, and −32 mV, respectively. These properties were stable for at least five weeks at room temperature. Furthermore, in vitro chrysin bioactivities regarding antioxidant and anti-AD were maintained as pure chrysin, suggesting that multistep formulation could not affect chrysin properties. Interestingly, the developed chrysin-NE was more tolerant of gastrointestinal digestion and significantly absorbed by the human intestinal cells (Caco-2) than pure chrysin. These findings demonstrate that the encapsulation of chrysin using oil-in-water nanoemulsion could enhance the bioaccessibility of chrysin, which might be subsequently applied to food and nutraceutical industries.