Recently, synthetic biology facilitates the engineering genetic circuits of microbes such as E. coli for the production of valuable bio-refineries. However, screening the large-size mutant library in a high-throughput manner has remained a challenge. Here, we describe a microfluidic bioreactor array that not only enables the high-throughput screening of a synthetically engineered mutant library but also extracts only target microbial cells using a hatch-like isolation method. The bioreactor array consists of the injection Layer and culture membrane, in which 16,000 microchambers were fabricated in an area of 4 cm×5 cm. Each microchambers were well compartmentalized for separate cell cultivation in a fed-batch mode. After screening, the microchambers containing the target cells with a mutant library punched out with a microneedle. A rich medium can be supplied through the holes so that only the target cells can further grow and come out of the microchambers. The automated extractor supports to deal with an even larger size of a mutant library. It causes no hazard to the cells than UV-curing, laser extraction and guarantees high-selectivity with high spatial resolution (0.05 μm). We ensure that the microfluidic bioreactor array will be broadly used for providing a novel means for mutant library analysis, drug screening, and cell sorting.