The selection of the appropriate fragment of the cell surface receptors as an antigen is significant for the production of antibodies. CD133, as a suitable biomarker candidate in the cancer stem cells (CSCs), is a glycosylated protein. The antibodies used for analyzing it recognize glycosylated epitopes of CD133. Since the glycosylated motifs have a dynamic nature over the lifetime of a protein, they limit the detection of CD133. In this study, to access a specific antibody against the antigenic, accessible, and non-glycosylated fragment of the native CD133, we performed an analysis. Then, we expressed the third domain (D-EC3) (serine641-leucine710) in BL21 (DE3), then the purified recombinant antigen immunized BALB/c mice. Finally, the dignity of an epitope of pure recombinant antigen has been approved by the interactions of antibody and antigen with the use of mice immunized sera via ELISA and flow cytometry experimentation. The results showed that the selected non-glycosylated fragment can compete well with the commercial antibody against the glycosylated epitopes to identify the native cell surface markers. The results can be considered for diagnosis and target therapy development of CD133+ cancer cells.