Activation of the NLR-family pyrin domain-containing 3 (NLRP3) inflammasome has been associated with diabetic retinopathy progression. We have synthesized a highly sensitive molecular imaging probe, InflammaProbe-2, as an early detection diagnostic tool for the in vivo molecular imaging of NLRP3 inflammasomes in living diabetic retina. The ability of InflammaProbe-2 for the targeted visualization of the NLRP3 inflammasome was assessed using an enzyme-linked immunosorbent assay (ELISA) by comparing its ability to inhibit the NLRP3-mediated secretion of IL-1β. InflammaProbe-2 was able to visualize NLRP3 inflammasomes in ARPE-19 cells treated under hyperglycemia as well as inflammatory conditions. Furthermore, InflammaProbe-2-dependent in vivo and ex vivo imaging of the NLRP3 inflammasome was achieved via fluorescence enhancement in streptozotocin (STZ)-induced diabetic retinopathy. In addition, the toxicity of InflammaProbe-2 was assessed in vitro and in vivo studies. InflammaProbe-2 showed no significant changes in the a-wave and b-wave amplitudes of their electrical response to flashes of light.