Chitosanases have received much attention because of their wide range of applications. Although most fungal chitosanases use sugar as their major carbon source, in the present work, a chitosanase was induced from a squid pen powder (SPP)-containing Penicillium janthinellum D4 medium and purified by ammonium sulphate precipitation and combined column chromatography. The purified D4 chitosanase exhibited optimum activity at pH 7–9, 60 °C and was stable at pH 7–11, 25–50 °C. The D4 chitosanase that was used for chitooligomers preparation was studied. The enzyme products revealed various chitooligomers with different degrees of polymerisation (DP) from 3 to 9, as determined by a MALDI-TOF mass spectrometer, confirming the endo-type nature of the D4 chitosanase. D4 chitosanase activity was significantly inhibited by Cu2+, Mn2+, and EDTA. However, Fe2+ activated or inhibited D4 chitosanases at different concentrations. The D4 chitosanase was also activated by some small synthetic boron-containing molecules with boronate ester side chains.