Cathepsin B (CTSB), an important lysosomal cysteine protease, plays a role in various physiological and pathological processes, and has an important effect on the ability of oocytes maturation and embryos development. Studying on the change regularity of CTSB activity of sheep oocytes during in vitro maturation (IVM), and the relativity with developmental capacity of oocytes, could provide a theoretical basis and technical measures to improve the efficiency of sheep embryo production in vitro. The amplified CDS sequence of CTSB from sheep, a total length of 1008 bp, coding 335 amino acids, had been submitted to Genbank (accession number: KC699918). Using a variety of bioinformatics software predicted that sheep CTSB protein had the typical structural characteristics of papain-like cysteine proteases, consisting of signal peptide, propeptide and mature peptide. It had three cysteine protease active sites: Cys108, His278 and Asn298, and also a CTSB protein specific characteristic “occluding loop”. E-64 also had modification of phosphorylation and glycosylation after translation. This experiment measured the CTSB activity change regulation in sheep cumulus oocyte complexes (COCs) during IVM using microplate reader by the fluorescent substrate method. The results indicated that the CTSB activity of sheep COCs showed a declining trend with the extension of IVM time. Comparing with the RFU ratio (1.000) of CTSB activity of adult sheep at IVM 0 h, it dropped to 0.7740 at IVM 8 h (P<0.05), 0.7030 at IVM 16 h (P<0.01) and 0.3709 at IVM 24 h (P<0.01). In juvenile sheep group, compared with the RFU ratio at IVM 0 h (1.0228), there was no significantly change at IVM 8 h (1.0267) (P>0.05), but it dropped to 0.7817 at IVM 16 h (P<0.01), 0.6087 at IVM 24 h (P<0.01). At IVM 8 h and 24 h, the CTSB activity of juvenile sheep COCs was significantly higher than adult sheep. Density mean of CTSB activity in sheep oocytes and cumulus cells were tested using laser scanning confocal microscope. The results showed that compared with the density mean of CTSB activity in oocytes at IVM 0 h (0.0119), it was extremely significantly decreased at IVM 24 h (0.0046) (P<0.01). And CTSB activity in cumulus cells at IVM 24 h (0.2175) was significantly decreased than IVM 0 h (0.2960) (P<0.05). The density mean of CTSB activity in oocytes and cumulus cells of poor quality group (0.0439 and 0.4371) were extremely significantly higher than the normal quality group after IVM 24 h (P<0.01).