The aim of the study was to create a self-emulsifying drug delivery system (SEDDS) with mucolytic properties based on incorporated papain for improved mucus permeation. In order to increase the lipophilicity of the enzyme and to dissolve it in SEDDS, hydrophobic ion pairing with sodium deoxycholate in a molar ratio of 20:1 (surfactant: enzyme) was performed. The yield of precipitated papain was 86.8 ± 2.7% and the ion pair was loaded into the formulations to 1% (m/m). Suitable formulations were chosen according to their properties to dissolve the ion pair and characterized regarding droplet size and polydispersity index. Prepared emulsions were in a droplet size range between 50 and 120 nm. Enzyme activity assay of complex and loaded SEDDS was conducted to ensure proteolytic qualities for following permeation and diffusion studies. SEDDS loaded with the ion pair showed an almost 2-fold increase in mucus permeation compared to the control without complex. Furthermore, 3-fold enhanced mucus diffusion could be confirmed in a second assay and an increase of mucosal residence on porcine intestinal mucosa up to 3- and 5-fold was observed as against the blank formulations. Consequently the incorporation of enzymes exhibiting proteolytic properties in self-emulsifying drug delivery systems may be considered as a promising strategy to enhance mucus permeation and overcome intestinal barriers.