Background Brilliant cresyl blue (BCB) staining can stain oocytes and differentiated oocytes will lead to different developmental outcomes. This technique has been studied in multiple species, but it is still unclear whether buffalo oocytes can be used for developmental potential prediction through BCB staining methods. This study used the BCB staining method to group buffalo oocytes (BCB + and BCB-) and perform in vitro maturation, in vitro fertilization, and embryo culture. By statistical analysis, the effect of BCB staining on predicting the developmental potential of buffalo oocytes will be explored. At the same time, molecular biology techniques will be used to detect gap junction protein and oxidative stress-related indicators to explore the molecular mechanism of BCB staining predicting oocyte cell developmental potential. Methods The oocytes were divided into BCB + and BCB- groups using BCB staining technique. And then mainly uses in vitro maturation, in vitro fertilization and embryo culture techniques of buffalo oocytes to analyze their developmental potential, and uses immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels, and qPCR to detect the expression levels of the antioxidant related genes SOD2 and GPX1. Results Our results showed that the in vitro maturation rate, embryo cleavage rate, and blastocyst rate of buffalo oocytes in the BCB + group were significantly higher than those in the BCB- group and the control group (P < 0.05). The expression level of CX43 protein in the BCB + group was higher than that in the BCB- group both before and after maturation (P < 0.05). The intensity of ROS in the BCB + group was significantly lower than that in the BCB- group (P < 0.05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB + group were significantly higher than those in the BCB- group (P < 0.05). Conclusions BCB staining can effectively predict the developmental potential of buffalo oocytes. The results of BCB staining are positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the ROS level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes may be closely related to antioxidant activity.