In this study, we describe the formation method of web-like three-dimensional (3-D) titania nanofibrous structures coated on transparent substrate via a high intensity laser induced reverse transfer (HILIRT) process. First, we demonstrate the mechanism of ablation and deposition of Ti on the glass substrates using multiple picosecond laser pulses at ambient air in an explicit analytical form and compare the theoretical results with the experimental results of generated nanofibers. We then examine the performance of the developed glass samples coated by titania nanofibrous structures at varied laser pulse durations by electron microscopy and characterization methods. We follow this by exploring the response of human bone-derived mesenchymal stem cells (BMSCs) with the specimens, using a wide range of in-vitro analyses including MTS assay (colorimetric method for assessing cell metabolic activity), immunocytochemistry, mineralization, ion release examination, gene expression analysis, and protein adsorption and absorption analysis. Our results from the quantitative and qualitative analyses show a significant biocompatibility improvement in the laser treated samples compared to untreated substrates. By decreasing the pulse duration, more titania nanofibers with denser structures can be generated during the HILIRT technique. The findings also suggest that the density of nanostructures and concentration of coated nanofibers play critical roles in the bioreactivity properties of the treated samples, which results in early osteogenic differentiation of BMSCs.