NLR family member X1 (NLRX1) is an important pattern recognition receptor that regulates innate immune response. In this study, coding region sequence and 1-492 bp N-terminal truncated sequence of chicken NLRX1 gene were firstly cloned by PCR and inserted into prokaryotic expression pET-32a (+) vector. Expression of the recombinant NLRX1 protein and N-terminal truncated protein (rNLRX1-part) were induced by isopropyl β-D-Thiogalactoside (IPTG). Moreover, the recombinant rNLRX1-part protein was purified and immunized a New Zealand rabbit. The titer of anti-NLRX1 polyclonal antibody prepared by indirect enzyme-linked immunosorbent assay (ELISA) was 1∶512 000. The Western-blotting analysis showed the prepared antibody could not only specifically react with NLRX1 protein from prokaryotic expression but can effectively detect NLRX1 over-expression in chicken DF-1 cells.