Nanobodies, also known as VHHs, originate from the serum of Camelidae. Nanobodies have considerable advantages over conventional antibodies, including smaller size, more modifiable, and deeper tissue penetration, making them promising tools for immunotherapy and antibody-drug development. A high-throughput nanobody screening platform is critical to the rapid development of nanobodies. To date, droplet-based microfluidic systems have exhibited improved performance compared to the traditional phage display technology in terms of time and throughput. In realistic situations, however, it is difficult to directly apply the technology to the screening of nanobodies. Requirements of plasma cell enrichment and high cell viability, as well as a lack of related commercial reagents, are leading causes for impeding the development of novel methods. We overcame these obstacles by constructing a eukaryotic display system that secretes nanobodies utilizing homologous recombination and eukaryotic transformation technologies, and the significant advantages are that it is independent of primary cell viability and it does not require plasma cell enrichment in advance. Next, a signal capture system of "SA-beads + Biotin-antigen + nanobody-6 × His + fluorescence-labeled anti-6 × His (secondary antibody)" was designed for precise localization of the eukaryotic-expressed nanobodies in a droplet. Based on this innovation, we screened 293T cells expressing anti-PD-L1 nanobodies with a high positive rate of targeted cells (up to 99.8%). Then, single-cell transcriptomic profiling uncovered the intercellular heterogeneity and BCR sequence of target cells at a single-cell level. The complete complementarity determining region (CDR3) structure was obtained, which was totally consistent with the BCR reference. This study expanded the linkage between microfluidic technology and nanobody applications and also showed potential to accelerate the rapid transformation of nanobodies in the large-scale market.