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Fluorescein angiography (FA) is an important tool for the diagnosis and management of diabetic retinopathy. However, the safety of fluorescein sodium on renal functions is not fully understood. One hundred type 2 diabetes patients, within the Ophthalmology Outpatient Clinic at Alexandria Main University Hospital, Egypt, were enrolled in this prospective observational study to determine the safety of FA on renal function. Serum creatinine and cystatin C were measured pre- and 2 days post-FA. Urinary neutrophil gelatinase-associated lipocalin (uNGAL) was measured pre- and 4 hours post-FA. Renal injury was defined as a 25% increase in serum creatinine, cystatin C, or uNGAL. The study included 71 females and 29 males, with a mean age of 55.73 ± 7.29 years. Baseline serum cystatin C and uNGAL were 0.89 ± 0.34 mg/L and 21.7 ± 2.39 ng/mL, respectively. Serum cystatin C and uNGAL significantly increased after FA to 0.95 ± 0.36 and 27 ± 2.81, respectively (P <0.001). Eleven patients (11%) experienced more than a 25% rise in serum cystatin C from baseline, whereas 40 patients (40%) experienced more than a 25% increase in uNGAL levels after FA. However, the mean serum creatinine level did not change significantly after FA (P = 0.061). Only one patient experienced more than a 25% rise in serum creatinine from baseline. FA showed a significant increase in early sensitive acute kidney injury biomarkers (as serum cystatin C and uNGAL) in substantial number of patients, suggesting but still not proving, a potential harmful effect of FA on kidney functions. These findings were not demonstrated using ordinary serum creatinine.