Ultrafine fibers were successfully fabricated from Channel catfish skin-extracted gelatin via electrospinning (ES). Important ES parameters, such as concentration of aqueous acid and fish gelatin solutions, and electric field intensity were examined to investigate the effects on the morphology of the gelatin nanofibers. Due to the poor mechanical properties of the fish gelatin membranes, composite nanofibers made of fish gelatin and poly(L-lactide)(PLLA) were produced with a novel solution. The introduction of PLLA remarkably improved the mechanical properties of the gelatin membranes. With a combination of good biocompatibility and mechanical properties, fish gelatin/PLLA blending non-woven mats are considered to be very promising in tissue regeneration area.