Vitamin A is essential for growth and differentiation of a number of cells and tissues, as result the precursor, a carotenoid β-carotene remains their essential source of vitamin A. However, the major problem this carotenoid face, is its susceptible to photodegradation and chemical oxidation, those properties make it difficult to use as an ingredient as functional food product and reduce its bioavailability. This study presents a novel approach to prepare a one-step inclusion complex using amylose microparticles as host molecule using amylosucrase from Deinococcus geothermalis (DGAS) and β-carotene nanoparticles, which were added into the DGAS enzyme reaction solution to entrap them during the synthesis of amylose chains. HRFE-SEM showing a spherical shape and average diameter of 4-8 μm; XRD and DSC showed an amorphous structure as well as less energy required to start the gelatinization process, due to the complexation of amylose chains with the β-carotene nano dispersion. Last but not least Raman spectroscopy was performed in order to confirmed the complex formation between β-carotene and amylose microparticles, showing the characteristic peaks of both compound. The stability test in this study showed the high stability of the complexed microparticles against environmental stresses such as, photodegradation and chemical oxidation. We expect this study contributes to the development of functional food materials to enhance the stability and bioavailability of active compounds.