This study was conducted to evaluate the relationship between serum insulin levels and the production of insulin antibody (IA) in type 2 diabetes (T2DM). A total of 647 T2DM were included. Among them, 20.9% patients were IA positive, who were elder and had a longer duration, lower BMI, a higher positive rate of glutamic acid decarboxylase antibody(GADAb) and higher serum insulin levels during an insulin secretion test. More patients were treated with insulin in IA positive group than in IA negative group (65.9% vs 41.0%, P=0.000). Fasting serum insulin level was associated with occurrence of IA in all patients (OR=1.02, P=0.001) and insulin treated patients (OR=1.033, P=0.002). The cut-off point of fasting serum insulin level for predicting IA positive was 17.87 mIU/L (sensitivity 55.1%, specificity 89.0%). Exogenous insulin use is associated with the presence of IA. Fasting serum insulin level can be used as a predictor for the production of IA in insulin-treated patients.