Ancient DNA (aDNA) analysis was employed to determine the sex of slave sacrifice victims from Qin State tombs during the Spring and Autumn Period of China. It is difficult to obtain sex information from fragmentary skeleton samples with the aid of skeletal morphology methods. aDNA was extracted from the dentine in selected tooth samples of sacrificial slaves using a modified traditional method, which combines together phenol/chloroform extraction, silicon dioxide adsorption and ultrafiltration concentration. Based on the sequence differences between the amelogenin homologous gene in the X and that in the Y chromosome, a pair of specific primers was designed to identify the sex of the selected samples. In the selected eight typical samples, the aDNA analytical results revealed that three were males and two were females. These findings indicate that molecular sex identification might provide more valuable information for archaeological research on the institution of slave sacrifice in the Spring and Autumn Period of China.