Methyl farnesoate (MF) is a sesquiterpene compound and the crustacean homolog of insect juvenile hormones. MF has multiple physiological functions involving the regulation of molting, reproduction, metamorphogenesis, behavior, and osmoregulation. In this study, the hemolymph levels of MF during ovarian development of Portunus trituberculatus were measured by gas chromatography-mass spectrometry (GC-MS). The results showed that the hemolymph level of MF in P. trituberculatus was low during stages I and II, increased considerably in stage III, and remained high in stage IV. Correlation of MF level with ovarian maturation indicates the putative stimulatory role of MF in this physiological process. As 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and farnesoic acid O-methyltransferase (FAMeT) are two essential enzymes in MF biosynthesis, their transcript levels during ovarian development were detected by quantitative real-time PCR (qPCR). Transcript levels of HMGR and FAMeT exhibited variation trends similar to that of the level of MF in hemolymph. This might indicate that high expression of HMGR and FAMeT could result in an increase in the production of MF, which ultimately affects ovarian development.