The aim of our study was to prepare and characterize chitosan-based nanoparticles encapsulating propolis extract by ionotropic gelation and glutaraldehyde cross-linking technique. Both spectroscopic (UV-Vis, FTIR) and microscopic techniques (AFM) were applied for structural characterization of nanoparticles, along with entrapment and release study of propolis extract. The physico-chemical properties and morphological features of the obtained nanoparticles demonstrated a good correlation between all the investigated methods. Moreover, the bioactive compounds were stable upon the encapsulation procedure. Propolis release from the polymeric matrix was monitored in both simulated gastric acid and simulated intestinal fluids, concluding that our proposed formulation is suitable for controlled release. These results may provide a novel design, with improved bioavailability, stability and nutritional value of propolis bioactive compounds during processing and storage, with possible applications in food and nutraceutical industries.