To evaluate the effect of third eyelid removal on the ocular surface of dogs, we operated on five young Beagle dogs and observed changes to tear function using the following tests: phenol red thread test (PRT), Schirmer tear test (STT-1), modified Schirmer tear test (STT-2), pH and tear break-up time (BUT). There was a significant decrease (37%) in STT-2 within 2 weeks after the excision and this declined further to 60% at 1 year. The pH value increased after excision. Presurgical pH was 7.17 ± 0.20 (mean ± SD), which increased to 7.55 ± 0.24 in the 14–60 days following removal, and further increased to 7.77 ± 0.65 at 1 year. The PRT and STT-1 decreased by 26% within 3–7 months compared to pre-excision values, but by 1 year the values recovered to near normal. The BUT pre-excision value was 24.0 ± 8.1 s, which shortened to 13.5 ± 4.5 s after 5 months and continued to decrease further during the study. There were no overt visual signs of KCS during the observational period. However, microinjury of the keratoconjunctival epithelium was observed for all operated eyes when vital staining was used at 1 year post surgery. Surgical excision of the third eyelid in Beagle dogs influenced tear quality level and affected the stability of the tear layer, and at 1 year there was evidence of microinjury to the keratoconjunctival epithelium.