To separate the fermentation products in real time and improve the fermentation yield, a novel poly-4-vinylpyridine-vinylbenzeneboronic acid polymer microspheres (p4VP-VPBA) with bifunctional groups was prepared by suspension polymerization. The spherical p4VP-VPBA was 5–20 μm and thermally stable below 355.4 °C. The elemental contents of N and O were 1.36% and 5.72%, respectively, and the content of boronic acid was 0.1357 mmol/g. Its adsorption performance to lactic acid was satisfactory. Under pH 4 and 20 °C, the maximum static adsorption of p4VP-VPBA was up to 185.31 mg/g in 3 h. Its selectivity on lactic acid to glucose was 70.05 at pH 2, 7.71 at pH 4, and only 0.81 at pH 10, which indicated a good selectivity to lactic acid at low pH fitting to fermentation process. Furthermore, a p4VP-VPBA-combined with hollow fiber membrane (HFM) separation system was proposed and the removal rate of 5 mg/mL lactic acid was maintained at about 90% after 5 consecutive cycles. These data showed a huge potential for future real-time capturing lactic acid from fermentation, which also was a reference for other fermentation inhibited by product concentration.