Abstract: Toll like receptors known as pathogen-associated molecular pattern (PAMPs) recognize microbe-specific immune elicitors and play fundamental roles in pathogen recognition and the activation of innate immunity. To better understand the functions of PtToll-1 in Portunus trituberculatus, the ectodomain of PtToll-1 was sequenced and subcloned into the expression vector, and the expressed recombinant ectodomain of PtToll-1 was obtained. The polyclonal antibody against rPtToll-1 was obtained from immunized mice using a conventional method. Results of SDS-PAGE showed that the recombinant protein had a molecular mass of 87.18 kD and it was predominantly expressed in the insoluble fraction. Moreover, western-blot analysis revealed that antiserum could specifically react with the rPtToll-1. The PtToll-1 were expressed in the tissues including intestine, hepatopancreas, gills, heart and muscle with high expression in gills and intestine. The PtToll-1 was mainly located in the connective tissue of digestive tract and gland, epithelial cells of the gill and hepatopancreatic tuble. Cell immunofluorescence and immunohistochemistry staining showed that PtToll-1 was mainly located in the membrane of the crab hemoctyes. Furthermore, the green fluorescence of the PtToll-1-GFP fusion protein was also located in the membrane of the HEK293 cells. These findings will help to better understand potential immune functions of the PtToll-1.