Fabrication of polycaprolactone (PCL)-chitosan (CS) three-dimensional (3D) scaffolds using the novel technique of melt stretching and multilayer deposition was introduced. In brief, firstly, the PCL-CS monofilaments containing 0% (pure PCL), 10%, 20% and 30% CS by weight were fabricated by melting and stretching processes. Secondly, the desired multilayer (3D) scaffolds were fabricated by arranging and depositing the filaments. Physical properties of the filaments and the scaffolds were evaluated. MC3T3-E1 cell lines were seeded on the scaffolds to assess their proliferation. A typical micro-groove pattern was found on the surfaces of pure PCL filaments due to stretching. The filaments of PCL-30%CS had the highest tendency of fracture during stretching and could not be used to form the scaffold. Increasing CS proportions tended to reduce the micro-groove pattern, surface roughness, tensile strength and elasticity of the filaments, whilst compressive strength of the PCL-CS scaffolds was not affected. The average pore size and porosity of the scaffolds were 536.90 ± 17.91 µm and 45.99 ± 2.8% respectively. Over 60 days, degradation of the scaffolds gradually increased (p > 0.05). The more CS containing scaffolds were found to increase in water uptake, but decrease in degradation rate. During the culture period, the growth of the cells in PCL-CS groups was significantly higher than in the pure PCL group (p < 0.05). On culture-day 21, the growth in the PCL-20%CS group was significantly higher than the other groups (p < 0.05). In conclusion, the PCL-20%CS scaffolds obtained the optimum results in terms of physical properties and cellular response.