The present study described the design and development of a new class of non-ionic vesicular self-aggregates and their utilization as glyco-responsive drug release vehicles. Cholesterol-based phenylboronic acid (PBA)-functionalized amphiphile was synthesized, and formed non-ionic spherical vesicular aggregates in aqueous milieu and hence was termed as a ‘niosome’. Various non-covalent forces involved in the formation of the niosome were thoroughly investigated by microscopic and spectroscopic techniques. The PBA group in the amphiphilic backbone rendered diol sensitivity in the niosome, due to which it showed morphological and physico-chemical changes in response to external glucose stimuli. Fascinatingly, the niosomes underwent dissolution upon the addition of glucose owing to the formation of a reversible boronate–diol adduct. This unique diol-sensitivity of the niosomes was utilized in the initiation of an enzymatic reaction using glucose as a switch. Here, the substrate entrapped within the niosome interacted with the enzyme in the presence of glucose as a consequence of the glucose-triggered dissipation of the niosome encapsulation, allowing accessibility of the substrate to the enzyme. However, in the absence of glucose, the enzymatic reaction failed as the enzyme could not achieve close proximity to the substrate. This glyco-responsive behaviour of the niosome was further employed in the field of tuneable drug release. Here, insulin was entrapped within the niosome without affecting the stability of the self-assembly. When external glucose was introduced to the drug–niosome conjugate, the niosomal encapsulation was destroyed, followed by release of the insulin. This unique glyco-sensitive drug-encapsulation efficacy and sustained drug release ability of the niosomal self-assembly make this soft-material highly promising in the development of cutting edge drug-delivery vehicles.