Intravitreal ranibizumab for diabetic macular oedema (DMO) has been recently shown to modulate levels of aqueous cytokines. This study investigates the associations between changes in aqueous cytokine levels following intravitreal ranibizumab therapy and the corresponding anatomical and functional changes in the eye. Twenty-five patients comprising 30 eyes diagnosed with DMO were prospectively recruited. All eyes received three loading dose ranibizumab injections at baseline, week 4 and week 8, followed by pro re nata treatment based on best-corrected visual acuity (BCVA) and central macular thickness (CMT) up to week 48. Prior to ranibizumab administration, aqueous samples were collected from all eyes, and subsequent sampling was performed at week 8. Levels of 32 cytokines were assessed at baseline and at week 8. At baseline, higher aqueous TNF-α levels were associated with poorer BCVA (p = 0.033), greater macular volume (p = 0.017) and worse diabetic retinopathy (p = 0.047). Higher levels of IL-7 were associated with poorer BCVA and greater macular volume (MV). Following treatment with ranibizumab there was a significant correlation with reduction of aqueous TNF-α and improvements in BCVA and MV, both at 6 months (BCVA [r = -0.558, p = 0.001], MV [r = 0.410, p = 0.024]) and 12-months (BCVA [r = -0.413, p = 0.023], MV [r = 0.482, p = 0.008]). The change in VEGF concentration following ranibizumab treatment did not correlate with either BCVA or MV improvements (p > 0.05). Higher levels of aqueous TNF-α and IL-7 correlated with worse DMO, both anatomically and functionally. Reductions in levels of aqueous TNF-α, but not VEGF, post ranibizumab treatment were associated with improvement in BCVA and MV.