CD133, a 120 KDa glycoprotein is a transmembrane glycoprotein which has been recently used as a cancer stem cell (CSCs) marker in a variety of carcinomas. CD133(+) cells possess strong tumorigenicity, responsible for tumor initiation and maintenance. Therefore, the goal of our study was to develop a novel CD133 humanized antibody as a promising target for cancer therapy. CD133 purified proteins were used for panning the naive human-semi-synthetic Tomlinson I + J phagemid library. The second extracellular domain (loop1) and the third extracellular domain (loop2) of CD133 were expressed in E. coli. In this study, we adopted a novel five-round selection strategy based on moderate stringent selection during the first rounds. This unique strategy was aimed at avoiding the loss of rare phages with high affinity to target proteins. After the five rounds of specific panning, six phage-antibody clones which specifically recognized recombinant human CD133 protein were obtained. The desirable phage clone named CD133-scFv-1 was cloned into the expression vector, then induced and purified. We show that CD133-scFv-1 and commercial murine antibody 293C3 could compete with each other in the indirect competitive immunoassay. Our work may lay the groundwork for future studies involving biological functions and applications of the CD133 humanized antibody.