Hybrid tissue engineered (HTE) scaffolds constituting polymeric nanofibers and biological tissues have attractive bio-mechanical properties. However, they suffer from small pore size due to dense overlapping nanofibers resulting in poor cellular infiltration. In this study, using nanosecond (ns) laser, we fabricated micro-scale features on Polycaprolactone (PCL)-Chitosan (CH) nanofiber layered bovine pericardium based Bio-Hybrid scaffold to achieve enhanced cellular adhesion and infiltration. The laser energy parameters such as fluence of 25 J/cm(2), 0.1 mm instep and 15 mark time were optimized to get structured microchannels on the Bio-Hybrid scaffolds. Laser irradiation time of 40 mu s along with these parameters resulted in microchannel width of similar to 50 mu m and spacing of similar to 35 mu m between adjacent lines. The biochemical, thermal, hydrophilic and uniaxial mechanical properties of the Bio-Hybrid scaffolds remained comparable after laser ablation reflecting extracellular matrix (ECM) stability. Human umbilical cord mesenchymal stem cells and mouse cardiac fibroblasts seeded on these laser-ablated Bio-Hybrid scaffolds exhibited biocompatibility and increased cellular adhesion in microchannels when compared to non-ablated Bio-Hybrid scaffolds. These findings suggest the feasibility to selectively ablate polymer layer in the HTE scaffolds without affecting their bio-mechanical properties and also describe a new approach to enhance cellular infiltration in the HTE scaffolds. (C) 2017 Elsevier B.V. All rights. reserved.