Cartilage degeneration is the major cause of disability and poses several challenges to repair and regenerate. Conventional surgical treatments often induce fibrous tissues and compromise its function. Alternative tissue engineering strategies utilized scaffolds, factors and cells alone or in combination with some degree of success. This study reports the use of nanostructured biomimetic scaffold system in regulating the rat bone marrow stem cells (rBMSCs) differentiation into chondrogenic lineage in vitro. The biometric scaffold is essentially a micro-porous polycaprolactone (PCL) spiral structure decorated with sparsely spaced bioactive PCL nanofibers. The bioactivity stems from the use of two major components of hyaline cartilage extracellular matrix (ECM) namely chondroitin sulfate (CS) and hyaluronic acid (HYA). The PCL spiral structure was surface functionalized with PCL nanofibers encapsulated with CS (20% (w/w)) and HYA (0.2% (w/w)). In order to retain and sustain the release of CS and HYA nanofibers were cross-linked using carbodiimide chemistry. This study also evaluated the effect of nanofiber alignment on rBMSCs differentiation and evaluated the production of characteristic hyaline cartilage proteins namely collagen type II and aggrecan in vitro up to 28 days. Rat bone marrow derived stem cells cultured on the aligned nanofibers expressed significantly elevated levels of collagen type II and aggrecan secretions (western blots) as compared to scaffolds decorated with random fibers and tissue culture polystyrene (TCPS). This fiber alignment dependent expression of collagen type II and aggrecan secretion were further confirmed through immunofluorescence staining. This biomimetic and bioactive scaffold may serve as a serve as an efficient scaffold system for cartilage regeneration.