Introduction Diabetic retinopathy (DR) is the leading cause of blindness in human and animal patients. Early detection and treatment of the disease are important and can be facilitated by proteomic approaches providing biomarkers. Material and Methods Tear films were collected on Schirmer strips from 32 canine patients (12 diabetic dogs without changes in the retina, 8 diabetic dogs with signs of DR, and 12 control dogs). Two-dimensional electrophoresis was used to separate tear film proteins prior to their identification with matrix-assisted laser desorption/ionisation–tandem time-of-flight mass spectrometry and interrogation of protein function databases to find matches. Results Five significantly differentially expressed proteins were identified; of those, one was downregulated (2ʹ-5ʹ-oligoadenylate synthase 3) and four were upregulated in the tear film of two diabetic groups (Ras-related protein RAB-13; aldo-keto-reductase family 1 member C3; 28S ribosomal protein S31, mitochondrial; and 60S ribosomal protein L5). The differentially expressed proteins identified in the tear film were involved in signalling pathways associated with impaired protein clearance, persistent inflammation and oxidative stress. Conclusion The results of our study confirm that the pathological process in the retina in the course of diabetes mellitus causes changes in the tear film proteome.