To further study the relationship between fish glucose metabolism and irisin, the irisin antibody and its application reliability need to be prepared and tested urgently. The Rosetta-irisin expression vector was constructed, and the mice were immunized after protein purification, dialysis, and ultrafiltration. The corresponding polyclonal antibodies were obtained and their specificity and antibody titers were detected. The common carp irisin detection method was established to detect the changes of irisinA and irisinB in carp serum after glucose tolerance and RNAi experiments. The immunofluorescence method was used to detect the expression of irisin in carp brain, heart, hepatopancreas, and intestine, and the changes of irisin content in these tissues after glucose tolerance. Detection of the irisin content after carp myocytes differentiation was conducted. Compared with the BL21-irisin expression vector, the expression of carp irisinA/B protein by the Rosetta-irisin expression vector increased by 2.3/1.6 times at 4 h of IPTG induction; irisinA and irisinB antibody titers were 9.0×10⁴ and 2.7×10⁵, respectively. And there was no cross-reaction between irisinA and irisinB polyclonal antibodies, the irisinA and irisinB can be measured respectively. IrisinA and irisinB contents showed different changes after OGTT. After RNAi, irisin was significantly decreased. Immunofluorescence results showed that irisin was the most abundant in the brain, followed by hepatopancreas, and relatively less in the heart and intestine. After OGTT, the fluorescence intensity of irisinA and irisinB in the brain, liver, heart, and gut increased significantly. The results of qPCR and immunofluorescence showed that the FNDC5 mRNA expression and irisin decreased significantly after carp myocyte differentiation. In this experiment, high affinity and specific irisinA and irisinB antibodies were prepared, and their application reliability was tested. The acquisition of this antibody laid the foundation for the systematic study of carp glucose metabolism. At the same time, the irisin detection methods could be widely used in the quantitative study of irisin protein levels in other fish.