In the current study, cloning and expression of the bovine coronavirus (BCV) nucleocapsid protein was carried out in a baculovirus expression system. The specific RT-PCR product of N gene was cut and extracted from gel using DNA gel extraction kit (Millipore). Eluted DNA was successfully cloned in pBlueBac4.5N5-His TOPO TA baculovirus transfer vector and transformed in chemically competent E. coli. A modified colony PCR assay was utilized to identify the positive bacterial colonies that harbor the recombinant plasmids carrying N gene in correct orientation. Generation of recombinant baculoviruses was achieved by co-transfection of Spodopetra frugiperda (Sf-9) insect cells with a linearized replication-defective baculovirus DNA (Bac-N-BlueTM) and the transfer vector. The recombinant baculoviruses were plaque purified; verified for the presence of target sequences using PCR and propagated for generation of high-titer viral stocks. In vitro expression studies utilizing the recombinant baculoviruses were conducted and revealed high-level expression of N protein as indicated by its distinct reactivity in immunofluoresence, solid phase ELISA and Western blot.