Proteomics analysis of sex-specific amelogenin peptides in tooth enamel has a high accuracy in determining sex, which is especially useful for poorly preserved fossil human remains. Destructive sampling has been used in most studies, which consumed a considerable been used in most studies, so less destructive analysis such as the one described here is more desirable. Since this method was developed, many researchers have used it to identify sex of archaeological samples. To verify and optimize this approach, the use of micro-destructive analysis of teeth with different preservation conditions is investigated using 24 samples from 11 sites (from Neolithic to the Qin-Han period). This method obtained sex identification results consistent with physical anthropology in all 18 archaeological samples with known sex. At the same time, six individuals of unknown sex were reliably judged, which shows that the micro-destructive analysis method has good applicability to tooth samples. We optimized this method in sampling and data analysis, noting the following points. 1) There is a certain linear relationship between number of total peptides and Ay-specific peptides. Combined with the fitting curve, 30 peptide fragments were identified as the reference threshold to exclude male false negative results. For samples without Ay-specific peptide segments, if the value was higher than 30, it was determined to be female; otherwise it’s sex was not determined. 2) Multiple extraction of peptides from HCl solution is an effective way to increase number and types of peptides without secondary damage to teeth. 3) Three kinds of variable modification (Oxidation (M) and Deamidation (NQ)) are necessary in database search. At the same time, the relationship between different factors and protein content was also evaluated. At present, there is no relationship between burial or thermal age, distribution area and preservation status of amelogenin peptides. How to characterize the preservation status of amelogenin in samples by appearance or other means remains to be further studied. The data obtained in this study also offers a research basis for further discussion of mechanisms affecting the preservation of amelogenin peptides. Therefore, experiments to evaluate preservation of enamel protein before proteomic analysis is an effective method to minimize the damage of samples.