Background: It remains a challenge to distinguish whether the damaged intestine is viable in treating acute mesenteric ischemia. In this study, photoacoustic imaging (PAI) was used to observe intestinal tissue viability after ischemia and reperfusion injury in rats.Methods: An in vivo study was conducted using forty male SD rats, which were randomly divided into a sham-operated (SO) group, a 1 h ischemia group, a 2 h ischemia group, and an ischemia-reperfusion (I/R) group with 10 rats in each group. In the ischemia group, the superior mesenteric artery (SMA) was isolated and clamped for 1 and 2 h, respectively, and in the I/R group, after ischemia for 1 h, the clamp was removed and reperfused for 1 h. The same time interval was used in the SO group. Immediately after establishing the animal model, a PAI examination was performed, and the small intestine was collected for histopathology.Results: The levels of PAI parameters Hb, HbR, MAP 760, and MAP 840 were increased to different degrees in the ischemia groups, especially in the 2 h ischemia group, compared with the SO group (P<0.05), and with prolongation of the ischemia time, the injury was aggravated. All PAI signal levels except HbO in the I/R group were higher than those in the control group, and the increased range differed, especially in Hb and MAP 840. Using western blot, compared with the SO group, the BAX increased significantly in the 2 h ischemia group (P<0.05), and Caspase-3 in the experimental group was significantly higher than in the SO group (P<0.05). The level of HIF-1α increased in the 2 h ischemia group and I/R group (P<0.05), and TUNEL staining showed that the number of positive apoptotic nuclei in the 2 h ischemia group was significantly higher than in the SO group (P<0.05). Hematoxylin-eosin (HE) staining showed that ischemia for 2 hours was the most serious, with obvious mucosal damage, extensive epithelial injury, and bleeding.Conclusions: PAI can be used as an effective tool to detect acute intestinal ischemia injury and quantitatively evaluate tissue viability.