Strain TKU024 was isolated from the soil in the middle Taiwan by using squid pen powder(SPP) as the sole carbon/nitrogen source and identified as Acinetobacter calcoaceticus. The optimized culture condition for chitosanase production was composed of 1.5% squid pen power(SPP), 0.1% K2HPO4 and 0.05% MgSO4．7H2O at pH 8 and incubated in 50 mL of liquid media in shaking flasks at 37℃ for 4 days. Two chitosanases(C1/C2) were purified from the culture supernatant by using ammonium sulfate precipitation, chromatography procedures of DEAE-Sepharose, Macro-Prep DEAE, and Phenyl Sepharose. The molecular mass of C1 and C2 determined by SDS-PAGE were approximately 27 kDa and 66 kDa, respectively. The results of peptide mass mapping indicated that C1 matched to chitinase from Cellvibrio japonicus Ueda107(GenBank accession number gi1192361966) with 4% sequence coverage and Pseudomonas aeruginosa PA01(GenBank accession number gi115596781) with 10% sequence coverage, and C2 matched to chitinase from Arthrobacter sp.(GenBank accession number gi127065204) with 7% sequence coverage. The optimum temperature, optimum pH, thermal stability, and pH stability of TKU024 chitosanase C1 and C2 were 50℃, pH 6,＜70℃, pH 8~9 and 60℃, pH 7,＜60℃, pH 6, respectively. C1 was inhibited by 5 mM Cu2+, Zn2+, Mn2+ and 2% (v/v) SDS, and was inhibited completely by 4~5 mM Mn2+ ; C2 was inhibited by 5 mM Ca2+, Urea, EDTA, Zn2+, Mn2+, 2% (v/v) SDS, Triton X-100, Tween 20 and 0.5% (v/v) Tween 20, but activated by 1 mM Mn2+.