Human amniotic epithelial cells (hAECs) have shown excellent efficacy in clinical research and have prospective applications in the treatment of many diseases. However, the properties of the hAECs and their proliferative mechanisms remain unclear. Here, single-cell RNA sequencing (scRNA-seq) is performed on hAECs obtained from amniotic tissues at different gestational ages and passages during in vitro culture. The results showed that the proliferation of hAECs is associated with epithelial-mesenchymal plasticity (EMP) during amniogenesis. Freshly isolated, full-term hAECs are identified as mature epithelial cells. Once cultured in vitro, they are observed to rapidly undergo epithelial-mesenchymal transition (EMT) and enter a partial epithelial-mesenchymal transition (pEMT) state to regain their EMP properties and proliferation capacities. With the continuous development of EMT, hAECs eventually enter a senescent state. The addition of SB431542 and microcarrier screening enabled the effective 3D expansion of hAECs by 50 fold while maintaining the EMP status in hAECs for further proliferation. This study not only elucidated the central proliferation mechanism of hAECs during development and expansion but also optimized the in vitro culture system so that it is sufficient to generate hAECs for 50 patients from a single donor amniotic membrane.