The lack of a PepT1 antibody for fish has hindered analysis of PepT1 protein expression by immune tissue chemistry or western blot. We analyzed the expression and distribution of PepT1 in L. at the transcriptional and protein levels. The immunogenic cDNA of PepT1 was obtained by PCR and the fragments were inserted into a pET-32a (+) Vector and transformed into Rosetta. The target polypeptide was expressed after induction with 1‰ IPTG. The molecular weight of the recombinant protein was measured by SDS-PAGE electrophoresis. The purified PepT1 recombinant protein was used to immunize New Zealand long-eared rabbits by ear vein injection combined with subcutaneous injection for 38 d to obtain rabbit anti carp PepT1 polyclonal antibody. Enzyme-Linked Immuno Sorbent Assay (ELISA) was used to evaluate the antibody titers, immunohistochemistry was used to check the tissue expression of PepT1, and real-time fluorescent quantitative PCR was used to evaluate the expression of PepT1 at the transcriptional level. The molecular weight of the target polypeptide was ~28 kDa, and the antibody titer was 4×10⁵, suggesting that activity was high. The PepT1 protein was expressed in the foregut, midgut, hindgut, spleen, hepatopancreas, and kidneys. The level of expression was remarkably higher in the foregut and midgut than in other tissues, which may be due to their roles in absorption of peptides during digestion. The positive immune staining region in the renal tissue was obvious and clear, and consistent with short peptides being re-absorbed by PepT1 distributed on the renal tubular basement membrane. Additionally, the PepT1 transporter was also expressed in the hepatopancreas and spleen, both metabolically active tissues in carp. In conclusion, the rabbit anti-carp PepT1 polyclonal antibody prepared in this study can effectively identify PepT1 from different tissues of carp. The expression pattern of PepT1 is similar to that at the transcriptional level. Our results provide a foundation for study of the structure and function of PepT1 at both the molecular and protein levels in carp. Additionally, we provide a basis for analysis of the relationship between small peptide absorption and protein metabolism. The antibody described here will be an important tool for localization and quantitative research of PepT1 in related fish such as , that belong to the family Cyprinidae or even in Cypriniformes.