The present invention aims to provide a tissue regeneration substrate excellent in penetrability to cells as well as capable of effectively preventing cell leakage from the tissue regeneration substrate to accelerate tissue regeneration； and a method of producing the tissue regeneration substrate. The present invention relates to a tissue regeneration substrate including: a nonwoven fabric made of a bioabsorbable material, the tissue regeneration substrate having a laminated structure in which a layer containing a nonwoven fabric having an average pore size of 20 to 50 µm and a layer containing a nonwoven fabric having an average pore size of 5 to 20 µm are integrated.
