Abstract: We optimized the two-dimensional electmphoresis technique for analyzing hemolymph in Penaeus monodon. To obtain high resolution 2-DE patterns, we examined the protein extraction, IPG-strip length, separation scope of IPG strip, sample loading amount and focusing condition by PDQuest software. Results indicate that PEG 6000 removed high abundance protein in haemolymph faster and more effectively, and increased the concentration of low abundance protein. The protein preparation with 80% TCA-acetone extraction treatment in isoelectric focusing was effective. The optimum 2-DE patterns in the proteins of Penaeus monodon were successfully separated by using pH 3~10 NL IPG strips with 18 cm length and 300 μg protein sample. The study provides stable and replicable method for relevant studies on hemolymph proteame of P.monodon and other similar crustaceans.