Nanoscale drug delivery systems devised from biocompatible and biodegradable molecules with functional groups which are readily amenable to external stimuli, have become a promising approach to circumvent the pitfalls associated with the hydrophobic drugs concomitant with the amplification of drugs’ action. Taking the cognizance of advantages offered by nanostructures and also in search of amphiphiles with intended biomedical applications, a novel and elite family of amphiphilic system that can self-assemble into enzyme-responsive supramolecular architectures has been synthesized via a chemo-enzymatic approach using an immobilized enzyme (lipase) from Candida antarctica (Novozym 435) and employing biocompatible starting materials, i.e. p-hydroxybenzoic acid, monomethoxypolyethyleneglycol (mPEG), and glycerol. The aggregation behavior of the resulting bolaamphiphiles have been studied using critical aggregation concentration and dynamic light scattering measurements, further supplemented by cryogenic transmission electron microscopic studies. The developed amphiphiles render efficient solubilization of model hydrophobic drugs and dyes, e.g. Nile red, nimodipine, and curcumin in aqueous solution. Moreover, the results demonstrate that a subtle structural modulation of the amphiphilic system alters the transportation behavior of the guest molecule. The investigation of the enzyme-responsive behavior of the synthesized bolaamphiphiles using a hydrolase enzyme, Candida antarctica lipase reveals that amide based nanocarriers disassemble and release the encapsulated cargo on incubation with the enzyme. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay unravels negligible cytotoxicity of the bolaamphiphiles at the tested concentrations, indicating their relevance in the development of nanocarriers for biomedical applications.