Objective:　To　isolate　and　purify　a　ribonuclease　from　Momordicacharantia　L.　and　study　the　enzymatic　and　biological　characterizations　of　the　ribonuclease.　Methods:　Ethanol　precipitation,　C18　reversed　phase　high　performance　liquid　chromatography　(RP-HPLC),　Tricine-SDS-polyacrylamide　gel　electrophoresis　and　isoelectric　focusing　were　used　for　isolation,　purication　and　identification　of　the　ribonuclease.　The　enzyme　activity　of　ribonuclease　was　detected　using　tRNA　from　yeast　as　substrate.　Results:　A　ribonuclease,　designated　MC　-RNase,　showed　a　high　purity　by　Tricine-SDS-PAGE　and　RP-HPLC.　Its　TV-terminal　sequence　was　determined　as　VNEFDYYQVVLQWQPAT.　The　molecular　weight　and　isoelectric　point,　was　estimated　to　be　14　ku　and　8.1.　Enzymic　experiment　showed　the　optimum　temperature　is　50,　and　optimum　pH　is　5,　Pb2+　can　increase　the　enzyme　activity,　while　Zn2+,　Fe2+,　Fe3+　and　Mg2+　inhibited　the　enzyme　activity　of　MC-RNase.　The　result　of　cytotoxicity　test　showed　MC-RNasehad　no　effect　on　the　proliferation　of　L-02　cells　and　Hep-G2　cells　in　vitro.　Conclusion:　Isolation　and　identification　of　a　novel　ribonuclease　from　fresh　Momordicacharantia　L.　©　2017,　Editorial　Office　of　Journal　of　CIFST.　All　right　reserved.