The cellular microenvironment can be engineered through the utilization of various nano-patterns and matrix-loaded bioactive molecules. In this study, a multilayer system of electrospun scaffold containing chitosan nanoparticles was introduced to overcome the common problems of instability and burst release of proteins from nanofibrous scaffolds. Bovine serum albumin (BSA)-loaded chitosan nanoparticles was fabricated based on ionic gelation interaction between chitosan and sodium tripolyphosphate. Suspension electrospinning was employed to fabricate poly-epsilon-caprolacton (PCL) containing protein-loaded chitosan nanoparticles with a core-shell structure. To obtain the desired scaffold mechanical properties with enough elasticity for expansion and contraction, a hybrid mono and multilayer electrospun scaffold was fabricated using PCL containing protein-loaded chitosan nanoparticles and poly-L-lactic acid (PLLA). According to the BSA release profile, the multi-layered structure of nanofibers with two barrier layers provided a programmable release pattern of the loaded protein. Moreover, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism spectra results showed that the electrospinning process had no significant effect on the primary and secondary structure of the protein. The results indicated a desirable biocompatibility and mechanical cues of the multilayer nanofibrous scaffolds supporting structural stability and controlled release of the protein, which can offer diverse applications in hollow organ tissue engineering. (C) 2015 Elsevier B.V. All rights reserved.