Abstract: The α -glucosidase gene malA was amplified by PCR from Enterobacter sakazakii (ATCC29544) genomic DNA. Then it was cloned into the expression vector pET22b (+) to acquire the recombinant expression plasmid pET22b-malA. It was transformed into Escherichia coli BL21 (DE3) to optimize expression. The fusion protein was purified by urea and separated by SDS -PAGE and recovered by gel extraction. White rabbits originate in New Zealand were immunized with the separated protein. The titer of polyclonal antibody was detected by ELISA and the binding capacity was identified by immunofluorescence assay. The result showed that the target gene was 1677bp and 100% identical to the corresponding gene in Genebank. SDS -PAGE analysis indicated that His -fusion protein was mostly lied in inclusion bodies, and the optimal expression condition was induced for 5h with 0.8mmol/L IPTG at the temprature of 37℃. The titer of polyclonal antibody was about 5 ×10 5 by ELISA. Immunofluorescence assay showed that the polyclonal antibody could bind E. sakazakii. These results would lay a foundation to prepare immunomagnetic beads for detection of E. sakazakii.