Endoinulinase production was achieved by heteroexpression of endoinulinase-encoding gene from Aspergillus ficuum which is an eukaryotic organism in Escherichia coli BL21 (DE3). Further analysis demonstrated that the native signal peptide existed in inu2 gene lowered the enzyme expression level. To realize extracellular accumulation of target protein and improve its expression level, native signal peptide was substituted with pelB, ompC, and pelB fusing with the native signal peptides; then, the effects on endoinulinase production were investigated. As a result, E. coli A606-3, with replacement of pelB as its signal peptide, showed the highest endoinulinase enzyme activity (75.22 U/mg). Also, it suggested that eukaryotic signal peptides have an inhibition on enzyme expression in prokaryotic organism. Moreover, the condition for inulooligosaccharide (IOS) production from inulin was optimized, and an IOS yield of 94.41 % was achieved under the condition of 15 % (w/v) inulin, purified endoinulinase dosage of 5 U/g inulin, 55 A degrees C, and pH 4.6 for 24 h. The major products of hydrolysis of inulin were identified as DP3 to DP7.