Phage display is one of the most frequently used platform technologies utilized to screen and select therapeutic antibodies, and has contributed to the development of more than 10 therapeutic antibodies used in the clinic. Despite advantages like efficiency and low cost, it has intrinsic technical limitations, such as the asymmetrical amplification of the library after each round of biopanning, which is regarded as a reason for it yielding a very limited number of antigen binders. In this study, we developed a high-throughput single-clonal screening system comprised of fluorescence immunoassays and a laser-driven clonal DNA retrieval system using microchip technology. Using this system, from a single-chain variable fragment (scFv) library displayed on phages with a complexity of 5.21 × 105 harboring random mutations at five amino acid residues, more than 70,000 clones-corresponding to ~14% of the library complexity-were screened, resulting in 78 antigen-reactive scFv sequences with mutations restricted to the randomized residues. Our results demonstrate that this system can significantly reduce the number of biopanning rounds, or even eliminate the need for this process for libraries with lower complexity, providing an opportunity to obtain more diverse clones from the library.