Polydimethylsiloxane (PDMS) microfluidic chips with various cross-junction angles were designed and fabricated by MEMS and soft lithography technologies to construct chitosan microfibers. Chitosan solution and sodium tripolyphosphate (STPP) solution were injected into the cross-junction microchannel of the microfluidic chip, which generated the laminar flow of chitosan solution by the hydrodynamic focusing. Cross-junction microchannels with angle 30°, 45° and 60° were designed to investigate the generation of laminar flow. The microchannel of angle 30° could produce smaller diameter of chitosan laminar flow than the other angles at the fixed flow rates. Different diameters of laminar flow from 15 μm to 40 μm were successfully generated and transformed into the chitosan microfibers using STPP solution, with the range of chitosan microfiber diameter from 20 μm to 50 μm. The schwann cells and fibroblast cells were employed for cell culture in chitosan microfibers, which would be applied in tissue engineering as scaffold.