Homo erectus remains have been found in Africa, Eurasia, and Southeast Asia, with a fossil record dating back to 2 million years, holding a significant place in human evolution. However, due to limited molecular evidence, its genetic characteristics, diversity, and potential connections to other archaic homonins and modern humans have long remained unresolved. To address these issues, a research team led by Qiaomei Fu from the Institute of Vertebrate Paleontology and Paleoanthropology of the Chinese Academy of Sciences, in collaboration with other archaeological institutions, successfully extracted ancient enamel proteins from six Middle Pleistocene H. erectus teeth (~0.4 Ma) from the Zhoukoudian, Hexian, and Sunjiadong sites. Further in-depth paleoproteomic analyses revealed the following breakthroughs. First, a quantitative sex determination pipeline was established based on male-specific amelogenin, Y isoform (AMELY), confirming that five of these specimens are male and one is female. Second, two genetically specific amino acid variants in ameloblastin (AMBN) were identified in all specimens from three sites. One is a newly discovered variant, AMBN-253G, which has not been found in any other archaic or modern human populations. It represents a molecular marker specific to the East Asian Middle Pleistocene H. erectus lineage, providing solid evidence that H. erectus specimens from Zhoukoudian, Hexian, and Sunjiadong sites belonged to the same evolutionary lineage. The other variant, AMBN-273V, has previously been identified in Denisovans. However, genetic analysis in this study reveals that this variant may have been introduced into Denisovans through populations related to these Middle Pleistocene H. erectus, and some of which subsequently contributed to certain modern human populations from Southeast Asia or Oceania. This study obtains lineage-specific molecular information from Homo erectus fossils for the first time, and reshapes our understanding of hominin evolution and the history of genetic admixture in East Asia.