Hybridomas are a commonly used, or even the only option, for laboratory study and pilot production of monoclonal antibodies (mAbs), which are crucial for both targeted therapy and biomedical study. A long-term culture of hybridomas will inevitably induce a heterogenization of the whole hybridoma population, resulting in a continuous growth of non-producing hybridomas. To overcome the limits of existing methods of screening heterogeneous hybridomas, in which the whole multi-round screening process is performed in multi-well plates or other discrete modules, this study presents a novel method in which all processing steps of a multi-round hybridoma screening are finished in a single microfluidic chip. This microfluidic chip comprehensively performs hybridoma trapping/proliferating/transferring and fluorescent identification of protein-antibody binding at single cell level. By performing a two-round screening of anti-CD45 mAb secreting hybridomas, the novel microfluidic chip was proved capable of screening several single high-producing hybridomas with minimum cell loss/human labor/time cost, and more importantly, enhanced accuracy and definite monoclonality, which is one of the most important properties of mAb production.