Currently, plant fibers, acquired from several resources, have been used widely for numerous applications by textile and green composite industries. The highlights of natural fibers include bio-degradable, uninterrupted source, easy and safe handling. The usage of enzymes in the textile industry fosters the development of eco-friendly technologies in fiber processing and tactics to improve the final product quality. The natural fiber from the stem of Cissus quadrangularis was extracted using an amylase enzyme. In light of these aspects, this work investigated the influence of enzyme usage on the extraction of natural fibers from the surface of C. quadrangularis stem through various characterization methods. The characterization of C. quadrangularis stem fibers (CQSFs) by Fourier transform-infrared spectroscopy, X-ray diffraction and thermogravimetric analysis were conducted. The surface morphology of the normal and fractured samples was studied using scanning electron microscopy. •It is a continuation work of Cissus quadrangularis fiber characterization.•Novel natural Cissus quadrangularis stem fiber enzyme treatment was characterized in this work.•Physical, chemical, thermal, and mechanical analyses were performed.•Characterized fiber has the potentiality to replace the harmful artificial fiber.•Cissus quadrangularis is available in plenty and has a potential for textile and green composite industrial applications.