The sterile alpha motif and HD domain 1 (SAMHD1) protein has been identified as a novel innate immunity restriction factor that participates in processes crucial to the viral life cycle. In the present study, we describe a procedure to generate monoclonal antibodies (MAbs) against porcine SAMHD1 and investigate its characteristics to analyze the expression of endogenous SAMHD1. The open reading frame of porcine SAMHD1 was cloned into the prokaryotic expression vector pCold-TF DNA to construct a recombinant plasmid pcold-pSAMHD1 and induce expression of recombinant porcine SAMHD1 protein by IPTG in Escherichia coli Rosetta. The purified recombinant porcine SAMHD1 protein was used to prepare MAbs of SAMHD1. After subcloning five times hybridoma cell clones expressing SAMHD1, MAbs were generated. Western blot analysis and indirect immunofluorescence assay showed that the overexpressed porcine SAMHD1 in 293T cells and endogenous SAMHD1 protein in porcine cell lines could be specifically recognized by the MAbs produced in this study. In conclusion, specific MAbs of porcine SAMHD1 are reported, and these MAbs provide a valuable tool for further studies of SAMHD1-mediated signaling in virus-infected cells to elucidate the underlying antiviral mechanism.