Aerobic vaginitis (AV) is a long-standing inflammatory disease that affects female patients. The use of antibiotics is a common means for AV treatment, but it will indiscriminately kill both pathogenic bacteria and beneficial strains, which easily causes vaginal dysbacteriosis and infection recurrence. Herein, we describe a bioinspired strategy for fabricating gelated cell sheet-supported lactobacillus biofilms (GCS-LBs) for AV treatment. Compared with common planktonic probiotic formulations, probiotic biofilms forming on a robust GCS exhibit enhanced stress tolerance and better colonization capacity in the mouse vagina. Moreover, DNA nanodevices are decorated on the GCS and dynamically report the microenvironment change of biofilms for timely evaluating bacterium activity, both in vitro and in vivo. Consequently, GCS-LBs are used for treating AV in an Escherichia coli-infected mouse model, which shows enhanced therapeutic efficacy compared with conventional antibiotic or lactobacillus monotherapy. Overall, the GCS-LB shows promise as a potent multifunctional tool to combat bacterial infection.