A new method for the separative determination of filamentous fungi amylase was described. The method is based on the measurement of the difference of calibration curve of amylase. The assay procedure was as follows : 1 ml of a enzyme solution diluted properly was incubated for 30 minutes at 37°C with 9 ml of the substrate solution (5 ml of 1% soluble starch solution and 4 ml of M/10-acetate buffer solution, pH 5.0). The enzyme reaction was interrupted by the addition of 1 ml of 1N-sodium hydroxide solution. After addition of 10 ml of Fehling's reagent, this reaction mixture was boiled for 2 minutes on direct fire, and cooled then to the reaction mixture 5 ml each of potassium iodide solution (30%) and diluted sulfuric acid (25%) was added. Iodine liberated hereby was titrated with N/20 sodium thiosulfate. (A) By using 6 times diluted enzyme solution of the previous enzyme solution, free reducing sugar was determined by the same manner (B). Unknown samples are then determined from coefficient table, which is prepared from experiments with the standard sample.