Ribozyme probe based on molecular beacon (MBR) for monitoring enzymatic cleavage process in real time is designed and studied. The approach relies on ri-bozyme substrates modified at the two arms, with a fluores-cent moiety attached to the end of one arm and a non-fluorescent quenching moiety attached to the end of the other arm. MBR is employed to directly convert the cleavage information into fluorescence signal in real time. Compared with traditional approach, this method provides a no-radiolabeling, sensitive and effective way to research on the ribozyme activity, enzymatic dynamic process and ri-bozyme function during gene therapy. The activity of the ribozyme against hepatitis C virus RNA (HCV-RNA) is stud-ied based on this assay. Ribozyme probe based on molecular beacon (MBR) for monitoring enzymatic cleavage process in real time is designed and studied. The approach relies on ri-bozyme substrates modified at the two arms, with a fluorescental moiety attached to the end of one arm and Compared with the traditional approach, this method provides a no-radiolabeling, sensitive and effective way to research on the ribozyme activity, enzymatic dynamic process and ri-bozyme function during gene therapy. The activity of the ribozyme against hepatitis C virus RNA (HCV-RNA) is stud-ied based on this assay.