Scaffolds comprising a nano- and micro-fibrous architecture are promising for tissue engineering, where nanofibers act as connecting network among microfibers and provide a 3D structural environment and mechanical stability for facilitating cell attachment, proliferation and migration. In this study, a novel structure was developed with polymeric micro and nano combined fibrous architecture, which aims to mimic the native extracellular matrix for tissue regeneration. Chitosan-tripolyphosphate (TPP) microfibers were prepared by wet spinning method, where collagen solution was allowed to self-assemble into nano/microfibers and subsequently freeze-dried for obtaining this combined architecture. To ensure prolonged mechanical stability, the scaffold was cross-linked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). This polymeric nano/micro combined scaffold revealed remarkable cellular activity and cytocompatibility towards both fibroblasts and osteoblast like cells and supported improved attachment and proliferation of cells than that of bare chitosan-TPP scaffolds owing to the presence of a bioactive molecule, collagen, in the intermingled form with chitosan-TPP microfiber. © 2012 Taylor & Francis Group, LLC.