Update information. Although the landscape of epigenomic and transcriptomic regulation during human pre-implantation development has been depicted by applying single-cell sequencing, the investigation of embryonic proteome is almost unrevealed due to the insufficient input quantity from precious human embryonic samples for traditional mass spectrometry (MS). With applying the state-of-the-art ultrahigh sensitivity mass spectrometry technology and nanoliter-scale oil-air-droplet (OAD) chip, we were able to identify more than three thousand proteins in a single oocytes during human pre-implantation development. Hundreds of stage-specific proteins with significant expressional changes were classified. Many that involve important functions, such as paternal genome reprogramming or DNA methylation, were firstly identified in human embryo studies. We discovered a two peaks of “zygotic proteome activation” (ZPA) at 2-cell and morula stages, in which proteins are mainly associated with epigenetic reprogramming. Our study, for the first time, delineates a comprehensive stage-specific proteome landscape at a single-cell level. It is as well a large step of enriching the panorama of functional genomics across human pre-implantation embryo development.