The study aimed to explore the correlation between retinal nerve fiber layer thickness (RNFLT) with blood biochemical indicators and cognitive dysfunction in patients with type 2 diabetes mellitus (T2DM) and the possible mechanism, thereby providing more theoretical basis for the occurrence and prevention of diabetes related complications. Eighty T2DM patients treated in our hospital from March 2022 to September 2022 were selected as the study subjects, and the clinical data of the patients were retrospectively analyzed. All patients underwent fundus fluorescein angiography (FFA) to analyze the changes in retinal blood vessels. Patients who met the inclusion criteria were divided as the diabetic retinopathy (DR) group (n=46) and simple diabetes group (n=34). The RNFLT, blood biochemical indexes and changes in cognitive functions of the patients were detected. The correlation between RNFLT with blood biochemical indexes and cognitive dysfunction was analyzed. Compared with the simple diabetes group, patients in the DR group had much lower mean, nasal, inferior and superior thicknesses (P<0.01). There existed no significant difference in blood pressure, body mass index (BMI), blood lipids (triglycerides, cholesterol, low-density lipoprotein, high-density lipoprotein) between the two groups (P>0.05). Compared with the simple diabetes group, patients in the DR group had much higher fasting blood glucose (FBG), hemoglobin A1c (HbA1c), fasting insulin (FINS), insulin resistance (HOMA-IR) index, apolipoprotein B (ApoB)/apolipoprotein A1 (ApoA1) (P<0.001). Besides, the DR group had sharply lower scores on the Mini-Mental State Examination (MMSE) scale and higher levels of the Trail Making Test-A (TMT-A) and TMT-B (P<0.001). Spearman correlation analysis confirmed that the mean RNFLT was negatively correlated with the levels of FBG, HbA1c, HOMA-IR index, TMT-A and TMT-B (P<0.05), positively correlated with the score of mini-mental state examination (MMSE) (P<0.05), and was no significant correlation with FINS and ApoB/ApoA1 (P>0.05). DR patients had significantly reduced RNFLT, elevated levels of blood glucose related indicators, and cognitive dysfunction. There existed a correlation between RNFLT and FBG, HbA1c, HOMA-IR index, TMT-A, TMT-B and MMSE.