Background: Determination of time passed since burn injury in the living is critical in forensic science. Autophagy biomarkers and vitronectin can play an important role in determination of the age of burn injuries through their levels in the tissue. Objective: The aim of this study was to investigate the role of autophagy biomarkers in dating burn injury and to correlate them with the histopathological effects of deep second-degree thermal burn. Method: Fifty-four male rats were used in this study after infliction of second-degree thermal burns to their skin. Samples were taken from them after 30 minutes and one, four, 24, 48, and 72 hours following burn to be examined histologically and also for autophagy biomarkers and vitronectin. Results: Significant reduction in the autophagy biomarkers (p < 0.001) over the first 24 hours then began to increase but still not reach the normal level up to 72 hours after burn. Vitronectin level increased after burn infliction 1.5-fold after first hour, then up to four-fold after four hours and after that began to decline but still did not reach the normal level up to 72 hours. Conclusion: Autophagy biomarkers can be used as a forensic tool in determination of the time passed since burn infliction in living.