Royal jelly has long been recognized as health food, with a high content of proteins. These proteins play important roles in honeybee caste and human health, but the proteomic analysis of those low-abundance proteins in royal jelly is always a challenge. Herein, we used the Osboren classification method to separate the royal jelly proteins of Xinjiang black bees, a sub-species of Apis mellifera mellifera, into various fractions. The globulin, ethanol-soluble protein and glutelin fractions were further separated by SDS-PAGE, and proteomic analysis was carried out by LC-MS/MS and searching against the NCBI database. A total of 63 proteins with definitive names were identified, in which 41 proteins were identified for the first time in royal jelly. The Osboren classification method combined with one-dimensional gel electrophoresis based proteomic analysis allows the identification of low-abundance proteins, and greatly extends the knowledge about the components and functions of royal jelly proteins.