Objective To investigate the feasibility of multiple-layers reconstruction of urethral tissue with 3D bioprinting technology. Methods Mixed blend of 7% GelMA and 2% Alginate with green and read microbeads respectively, or 2×106cells/mL urethral epidermal cells and smooth muscle cells, printed on the system of co-axial multiple channel bioprinting platform. The speed of inner and outer layer printing was 10-20 μL/min and CaCl2extruding was 80-200 μL/min. Then the printed structure was observed under microscopy, and the printed hollow tissues loaded urethral cells were tested with live/dead and F-actin staining from day 0 to day 7. The printed hollow structure was perfused with color medium after cultured 24 hours in vitro. Results With hydrogels mixed with microbeads, two distinct sublayers of printed hollow structure can be observed clearly under microscopy. The diameter of inner and outer layer was (663.66±51.74) μm and (976.84±63.43) μm respectively, and wall length of inner and outer layer was (62.06±10.45) μm and (93.78±10.25) μm respectively. The viability of printed urethral tissue was 84.52% and 86.26% at day 0 and day 7. F-actin expression were observed on day 7 after immunofluorescent staining and showed the spread cells. During the process of perfusion of hollow tissue, colored medium can circulate the channel smoothly without color exuding. Conclusion Urethral hollow structure can be printed with combined hydrogel by the method of co-axial multiple channel bioprinting system. And the urethral cells can survive for long term after printing.