Abstract: An electrophoresis separation layer is produced which comprises a polyacrylamide gel which remains stable even when stored for a long time and is available for analyzing substances of an extremely wide molecular weight range in electrophoresis for determining molecular weight. The electrophoresis separation layer of the present invention includes a polyacrylamide gel with an aqueous solution containing an amine of a specific pH value, an ampholyte and an acid at a specific ratio as an electrolytic solution in the gel. This separation layer can be produced by lowering the pH value of the electrolytic solution in the polyacrylamide gel to such an extent as not to cause any hydrolysis of amide while achieving a suitable potential gradient distribution in the gel during the electrophoresis.