Type 2 diabetes mellitus is associated with an increased risk for stroke and unfavorable outcomes following stroke. Matrix metalloproteinase-9 (MMP-9) is a potential contributor to the poor prognosis of diabetic ischemic stroke. Investigations on diabetic stroke are limited by the lack of non-invasive imaging techniques. In this study, we report a fast and ultra-sensitive MMP-activatable optical imaging probe (MMP-P12) to achieve non-invasive and real-time visualization of the dynamic expression of MMP-9 in diabetic stroke. Moreover, by using this probe, we aim to detect the therapeutic efficacy of CD28 SA in diabetic stroke. Serial near-infrared fluorescence (NIRF) imaging was performed on wild-type and STZ-induced diabetic mice after MMP-P12 probe injection on days 1, 3, and 7 post ischemic stroke. The dynamic change in MMP-9 expression after CD28 SA treatment was also imaged on days 1, 3, and 7 post stroke and confirmed by immunohistochemistry staining and western blotting. NIRF imaging showed that diabetic stroke mice presented a trend of higher levels of MMP-9. CD28 SA treatment significantly downregulated the expression of MMP-9 on day 7 post stroke. Glucose also had a downward trend in CD28 SA treated diabetic stroke mice. In conclusion, our data suggest that MMP-P12 probe successfully detect the dynamic change of MMP-9 in diabetic stroke by utilizing optical imaging. CD28 SA treatment decreased the expression of MMP-9 and could be a promising therapeutic strategy for the treatment of diabetic stroke.