Objective To design and fabrication of the 3D scaffolds that exhibit biomimetic multi-scale structures composed ofmicroporous structure with nanofibers for urethral tissue.Methods By analyzing themulti-scale structures of urethral acellularmatrix,a bilayer scaffold composed ofmicroporous section and nanofibrous dense section to biomimetic urethral tissue was designed.Template biosynthesis was used to fabricate 3D microporous nanofibrous gelatin/BC bilayer scaffolds(BC-Gel/BC)by stationary cultivation usingmicroporous gelatin scaffold as a template.Results The BC-Gel/BC bilayer scaffold composed of dense section of BC nanofibrils network and microporous section of Gel/BC.Themicroporous section with highly interconnected micropore(167±56)μm and surface decorated on themicropore wall by BC nanofibers (20-40)nm were fabricated,which were remarkably similar structure to the native extracellularmatrix(ECM).Conclusion Using the template biosynthesis method,the BC-Gel/BC bilayer scaffold for urethral tissue engineering can be successfully constructed,which has the bilayer structure,m icro porous and nanofibrous network to biom imetic urethral tissue.