Fasciola hepatica, a trematode helminth, causes an economically important disease (fasciolosis) in ruminants worldwide. Proteomic analysis of the parasite provides valuable information to understand the relationship between the parasite and its host. Previous studies have identified various parasite proteins, some of which are considered as vaccine candidates or important drug targets. However, the approximate distribution and abundance of the proteins on the surface and within internal parts of the liver fluke are unknown. In this study, two fractions including surface protein fraction (representing surface part of the parasite, near subplasma membrane of the tegument and above the basal membrane of the tegument) and internal protein fraction (representing internal part of the parasite, mainly deeper sides of the tegument including subbasal membrane and other further internal elements of the parasite) were obtained. Components of these two fractions were investigated by an advanced proteomics approach using a high-definition mass spectrometer with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanoUPLC-ESI-qTOF-MS). FABP1 was found highly abundant in the SPF fraction. Potentially novel F. hepatica proteins showing homology with AKT interacting protein (Xenopus tropicalis), sterol O-acyltransferase 2 (Homo sapiens), and integrin beta 7 (Mus musculus) were identified with high quantities in only the surface fraction of the parasite and may be possible candidates for future control strategies.