Simultaneous aqueous two-phase extraction and saponification reaction of chlorophyll from silkworm excrement was investigated in this study, and it was compared with the saponification reaction followed by aqueous two-phase extraction (ATPE) method. The results indicate that it is better to use the simultaneous aqueous two-phase extraction and saponification reaction method than other method. The effects of different parameters on the amount of sodium chlorophyllin in the top system of the ethanol NaOH aqueous two-phase system (ATPS) were studied, including salt concentration, saponification temperature and time. When the NaOH concentration was 0.4 g/ml, saponification temperature and time were 333.15 K and 2 h respectively, the amount of sodium chlorophyllin in the top system reached its maximum in the investigated range. Further, the top system of the ethanol NaOH ATPS was taken out and used to prepare sodium copper chlorophyllin, and the absorbance ratio (A(405.00nm)/A(627.00nm)) was 3.67. (C) 2013 Elsevier B.V. All rights reserved.