In this work, electrospun membranes of biodegradable polylactic acid and hydrosoluble collagen with multilevel architectures were fabricated by pattern template electrospunning method. The performances influenced by constituent of electrospun matrix and multilevel structures of electrospinning membranes were discussed. The mechanical properties and hydrophilicity were improved both by blending nature collagen and inducing the multilevel structures. The multilevel-architecture membranes were seeded by L929 fibroblast cells with phenotypic shape and guided growth according to pattern orientation. Cells infiltration through the fibers, cell attachment, and proliferation with fiber membranes were observed during the process. More interestingly, for further investigation of promotion the cell infiltration by multilevel structures, the electrospinning membranes were processed into tubular scaffold with 3D print scaffold. The in vivo result confirmed the scaffold favourable biocompatibility and improved tissue regeneration behaviors in vivo. The novel biodegradable membranes and scaffolds will be applicable for tissue engineering based upon their particularly multilevel architectures, which acts to support and guide cell growth in regular way.