In this study a gonadotropin-releasing hormone (GnRH)-like peptide was partially isolated by reverse phase high performance liquid chromatography (RP-HPLC) from the brain extract of Chinese mitten crab Eriocheir sinensis. Immunological dot blot analyses from RP-HPLC fractions using the anti-octGnRH revealed that one of fractions is immune reaction positive, suggesting that the fractions of brain extract contain GnRH-like substance. Eight kinds of small peptides, with molecular weight ranging from 800 to 1 400 u, were further identified in this fraction by using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS). One of peptides in MS component shared similar molecular weight with the octGnRH.