Diquafosol ophthalmic solution (DQS) stimulates P2Y2 receptors on the ocular surface, which enhances mucin secretion from goblet cells. Therefore, tear film stability and hydration of the ocular surface can be achieved independent from lacrimal gland function. This prospective, open-label pilot study included 60 eyes of 30 diabetic patients diagnosed with dry eye disease (DED) and were randomly assigned to either DQS ( = 30 eyes) or hyaluronate (HA) group ( = 30 eyes). Participants in the DQS group received 3% diquafosol ophthalmic solution, whereas HA group received 0.1% sodium HA preservative-free artificial tears. The dosage for both drugs was 1 drop, 6 times per day for 4 weeks. Tear film lipid layer (TFLL), noninvasive breakup time (NITBUT), corneoconjunctival staining (CS) score, meibomian gland (MG), conjunctival hyperemia [redness score (RS)], ocular surface disease index (OSDI) was assessed and compared at baseline, day 14, and day 28. Comparing baseline and day 28 measurements revealed that both groups found significant improvements in NITBUT, CS, MG quality, MG expressibility, and OSDI scores significantly ( < 0.05), in addition TFLL improvements were only found in the DQS group. At day 28, the magnitude of change in mean NITBUT was 1.74 (DQS) versus 0.31 (HA), 1.16 (DQS) versus 0.37 (HA) point grade reduction in corneoconjunctival staining score and 9.80 (DQS) versus 4.80 (HA) point grade in mean OSDI score. Three percent diquafosol ophthalmic solution treatment demonstrated the ability to improve the tear film dry eye parameters and clinically reduced sign and symptoms of DED in diabetic dry eye patients. Clinical Trials.gov ID: NCT04980144.