L-Asparaginase II from bacteria has been used in treatment of acute lymphoblastic leukemia. In this study, ASNase II gene from Citrobacter freundii1101 was sequenced and cloned in E.coli DH5α. For this purpose, three pairs of primers were designed to amplify different fragments of the gene. After sequencing ansB by DNAMAN software, the full length of the gene was amplified by PCR. ASNase II gene was cloned into the pET22b plasmid and transformed into E. coli BL21pLysS (DE3)-competent cells by the heat shock method. The expression of periplasmic recombinant ASNase II was induced via IPTG (1mM) in transformed E.coli cells. Periplasmic ASNase II was extracted using an alkaline lysis method. The extracted protein was purified by one-step DEAE-Sepharose fast-flow chromatography. ASNase II activity was considered as an index for the protein expression. Bioinformatics analysis of Citrobacter freundii1101- ASNase II gene showed some similarity with the therapeutic enzyme from E.coli K12 and Erwinia. BLAST analysis showed more than 91% and 99% similarity in nucleotide and amino acid sequences, respectively, with other strains of Citrobacters; and also a similarity of 82.52% and 92.82% in nucleotide sequence and amino acid sequences, respectively, with E.coli K12. The antigenicity of the protein was predicted and compared with the therapeutic enzyme using semi-empirical method. These results indicated other potent bacteria source of ASNase II as a candidate for anticancer consideration.