Fe₃O₄ nanoparticles (NPs) can be conjugated with acridine orange to create a hybrid nanomaterial with unique properties, such as the magnetic characteristics of magnetite and the fluorescence of acridine orange, making them useful for a variety of applications, including cell imaging, drug delivery, and magnetic separation. In this context, we aimed to provide a biological evaluation of this type of NPs using in vitro co-culture models of human skin, lung and intestine. Fe₃O₄ NPs were obtained by the co-precipitation method from Fe²⁺ and Fe³⁺ (1:2 molar ratio). The concentration of acridine orange in an aqueous NH₄OH solution was 0.00025%. The product was washed several times with ultrapure water, redispersed, and centrifuged thrice at 6000 × g for 10 minutes. Each supernatant was collected, obtaining three different suspensions of NPs. We developed one model of skin barrier using a co-culture of human keratinocytes (HaCaT cell line) and dermal fibroblasts (CCD-1070Sk cell line), one model of pulmonary barrier made of A549 epithelial cells and MRC-5 fibroblasts, and one model of intestinal barrier composed of human Caco-2 enterocytes and HT-29-MTX mucus-producing intestinal cells. Our results showed that none of the NP suspensions influenced the cell viability of the co-culture systems, suggesting their good biocompatibility on short-term exposure (24 hours) according to the cytotoxicity assays performed. In addition, we observed a specific apical-basal cell polarization in the co-culture systems, being maintained after one-day exposure to the three suspensions of NPs. To sum up, Fe₃O₄ NPs conjugated with acridine orange could be promising hybrid nanomaterial with good biocompatibility and special properties for future applications in biomedicine.