The invention relates to a dog rabies virus NP-ELISA antibody detection kit, wherein, an antibody detection plate is a detachable 96-hole enzyme labeled plate which is coated by rabies virus nucleoprotein NP, enzyme conjugate working liquid is a horseradish peroxidase labeled anti-dog IgG monoclonal antibody, a positive control is dog rabies standard positive serum, a negative control is a dog standard negative serum, the rabies virus nucleoprotein NP is a fragment of rabies virus ERA vaccine strain NP gene 1350bp which is amplified by RT-PCR technology, the rabies virus nucleoprotein NP is inserted into the downstream of a prokaryotic expression vector pET-28a to construct a pET-NP recombinant expression plasmid, Escherichia coli BL21 DE3 is converted by the calcium chloride method, 0.01mM IPTG induced expression is carried out, ultrasonic waves are used for the lysis of an expression strain, and the purification by a Ni-NTA affinity chromatography column is carried out for the preparation. The using concentration of the rabies virus nuclear NP recombinant protein for coating the NP antibody detection plate is 2 mug / ml, and the using amount is 100 mu 1 / hole. The dog rabies virus NP-ELISA antibody detection kit has the advantages of strong specificity, high sensitivity and good safety.