To study the association between the fluorescence levels on fluorescein angiography images and the characteristics on spectral-domain optical coherence tomography (SD OCT) images in diabetic macular edema (DME). Retrospective, observational, cross-sectional study. One hundred sixty-seven consecutive eyes of 116 patients with diabetic retinopathy for whom FA and SD OCT were performed on the same day. Fluorescein angiography using the Heidelberg Retina Angiograph 2 and OCT images using Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany) were obtained. The leakage of fluorescein dye in each subfield of the Early Treatment Diabetic Retinopathy Study (ETDRS) grid was quantified and defined as fluorescence levels, which were compared with the retinal thickness and foveal pathomorphologic features evaluated by SD OCT. The relationship between fluorescence levels and the foveal pathomorphologic features on SD OCT images. One hundred twelve (67%) eyes with center-involved DME had significantly higher fluorescence levels in all subfields of the ETDRS grid than 55 (33%) eyes without DME. Fluorescence levels were correlated modestly with the retinal thickness in individual subfields in eyes with center-involved DME. Thirty-seven eyes with foveal serous retinal detachment (SRD) had greater retinal thickness in all subfields and higher levels of fluorescence in most subfields, except the superior subfield of the inner ring. After adjusting for the central retinal thickness using multivariate analyses, eyes with SRD had significantly (P = 0.0085) higher fluorescence levels in the nasal subfield of the inner ring and the superior, nasal, and inferior subfields of the outer ring (P = 0.0117, P = 0.0020, and P = 0.0017, respectively). However, the fluorescence levels in any subfields of the inner or outer ring did not differ significantly between eyes with and without foveal cystoid spaces. The correlation between the fluorescence levels and retinal thickness suggests that the vascular hyperpermeability in the perifovea contributes to the pathogenesis of foveal SRD in DME.