A high-throughput screening system based on droplet microfluidic sorting was developed and employed for screening of high lactic acid-producing Bacillus coagulans. In this system, water-in-oil-in-water (W/O/W) droplets, which were ∼12 pL in volume were used as picoliter-reactors for lactic acid fermentation. A fluorescent sensor was developed and used for monitoring pH which indicated the production of lactic acid. After fermentation, fluorescence activated cell sorting was performed with high sensitivity and speed. Using this microfluidic high-throughput screening system, we found a mutant with a yield of 76 g L⁻¹ lactic acid which was 52% higher than its parent strain with a screening throughput exceeding 10⁶ clones per h.