Amniotic membrane has been widely applied as a biological graft in both medical and veterinary practice. In ophthalmology, epidermal growth factor (EGF) in human amniotic membrane (HAM) promotes corneal epithelial cell proliferation and migration, thus it facilitates corneal wound healing. In dogs, with limited cryopreserved HAM availability, different cold glycerol preserving protocols have been developed for the storage canine amniotic membrane (CAM). This study aimed to study protein expression of EGF in CAM preserved with different concentrations of glycerol and storage temperatures, using enzyme-linked immunosorbent assay. CAM preserved in 50% glycerol and 99.5% glycerol and kept at 4 and − 20 °C for 7–30 days were compared. We found that preserving membrane with 50% glycerol at − 20 °C has significantly higher EGF protein expression compared with that at 4 °C (p < 0.05). There was a trend that the storage in 50% glycerol achieved higher EGF protein expression than 99.5% glycerol at both 4 °C and − 20 °C. In conclusion, 50% glycerol at − 20 °C was the best condition to preserve CAM in our study. Therefore, there is likely an alternative method to maintain level of EGF protein expression in preserved CAM.