As a way to modify both the physical and biological properties of a highly elastic and biodegradable polyurethane (PU), silk fibroin (SF) with rigidity, biocompatibility and non-toxicity was blended with PU having SF/PU=1/3, 2/2 and 3/1 weight ratios. Bio-based high elastic PU was prepared from hexamethylene diisocyanate and isosorbide/polycarbonate diol (4/1) by simple one-shot bulk polymerization and nanofibers were electrospun directly from SF and PU blend. With increasing SF content, the tensile strength decreased as did the strain at break; the stiffness increased to around 150 MPa for the SF/PU (3/1) with highest silk content. On the other hand, SF/PU=1/3 blend nanofiber showed a 150 % strain at break. Degradation tests performed at 37 degrees C in phosphate buffer solution showed a mass loss of 30% after 4 weeks, which showed an initial rapid weight loss. The in vitro cytocompatibility test results following culture of C2C12 (a mouse myoblast cell line) on the nanofiber film surface showed that relative cell number on all of the blend films after 2 days was higher compared to the proliferation rate on the optimized tissue culture plastic. These polyurethanes offer significant promise due to soft, flexible and biocompatible properties for soft tissue augmentation and regeneration.