Background: Analyzing recovered evidence, such as blood which is one of the most encountered types of biological evidence, can provide information to establish the definite time when a crime was committed. This study aims to investigate the timeand temperature-related effects on human bloodstain’s α-globin messenger RNA expression and to estimate the bloodstain’s age using α-globin mRNA. Methods: A total of 22 blood samples were collected from healthy middle-aged volunteers (12 women and 10 men). After preparation, the samples were exposed to temperatures of 4°C, 24°C, and 40°C. Next, the mRNA expression of the α-globin gene was quantified by real-time RT-PCR at different time intervals of 0, 30, 90, and 150 days. Results: The α-globin gene expression showed the highest mean values by 0 day and at 4°C and the lowest mean values by 150 days and at 40°C. Samples from male participants showed higher mean values of α-globin gene expression compared to their female counterparts. A significant negative correlation was detected between α-globin gene expression and time interval. Meanwhile, a regression equation was formulated to estimate the time interval using the α-globin gene concentration. Conclusion: α-Globin mRNA could be a useful marker to estimate the age of human blood spots.