A molecular hyperspectral imaging (MHI) system was developed to evaluate the protective effects of erythropoietin (EPO) on early diabetic retinopathy in rats. The system was used to capture hyperspectral images of rat retinal sections selected from three groups: normal control, diabetic, and EPO. Three biochemical parameters were defined, namely, the spectral transmittance index, thickness of outer nuclear layer, and cell area percentage. The corresponding algorithms to calculate these were likewise presented. Experimental results show that, after treatment, the newly defined biochemical parameters of the EPO group become more similar to those of the normal control group compared with those of the diabetic group. This indicates that, to some degree, EPO provides protective effects on the retinal cells of chemically induced diabetic rats after it is injected intravitreally at the onset of diabetes. The results likewise show that the MHI system could provide useful quantitative information regarding retinal sections, which ophthalmologists can use to determine the pathogenesis of diabetic retinopathy and evaluate the protective effect of EPO on diabetic retinal cells.