Flow-assembled chitosan membranes are robust and semipermeable hydrogel structures formed in microfluidic devices that have been used for important applications such as gradient generation and studying cell-cell signaling. One challenge, however, remains unresolved. When a polydimethylsiloxane (PDMS) microchannel with a flow-assembled, deprotonated chitosan membrane (DCM) is treated with anti-adhesion agents such as Pluronic F-127 to prevent biomolecular and cellular adsorption on PDMS, the interaction between DCM and PDMS is compromised and the DCM easily delaminates. To address this challenge, DCMs in microfluidics are crosslinked with glutaraldehyde to modulate their properties, and the altered properties of the glutaraldehyde treated chitosan membrane (GTCM) are investigated. First, the GTCM's acidic resistance was confirmed, its mechanical robustness against hydrostatic pressure was significantly improved, and it remained intact on PDMS after Pluronic treatment. Second, crystallization in DCM and GTCM was investigated with quantitative polarized light microscopy (qPLM), which revealed that GTCM's optical retardance and anisotropy were lower, implying less molecular alignment than in DCM. Finally, membrane permeability was tested with FITC-labeled dextran transport experiments, which showed that the transport across GTCM was slightly higher than that across DCM. Overall, glutaraldehyde-crosslinked chitosan membrane has better acidic resistance, higher strength under Pluronic treatment, and less molecular microalignment, while its semi-permeability is retained. This study demonstrates how glutaraldehyde crosslinking can be used to modify and improve biopolymer membrane properties for broader applications, such as in an acidic environment or when Pluronic passivation is needed.