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Aim: To improve the long-term storage stability of a bioactive protein and the subsequent colon-targeted release, a double-layer chitosan (CS)-based particle was developed. Methods: To form the double-layer CS-based particle, the second layer was crosslinked onto the single-layer bovine serum albumin (BSA)-loaded CS-based particle. The structure and properties of particles were further investigated. Results: With the second layer, the double-layer particles became more compact, which was important for the inhibition of bioactive protein leakage during storage through strong electrostatic interactions and swelling of the hydrogels. After 30 d of storage, there was only 43.74–49.32% BSA leakage from the C15-TPP/C15-HMP double-layer particles. Moreover, the BSA release in subsequent colon-targeted delivery after storage was 44.02–48.59%. Conclusions: With double-layer shielding and a more compact arrangement, it was possible to reduce bioactive protein leakage over long periods storage and achieve subsequent colon-targeted delivery. [ABSTRACT FROM AUTHOR]