Bioelectrochemical sensors have proven attractive as simple and low-cost methods with high potential for online monitoring of volatile fatty acids (VFA) in the anaerobic digestion (AD) process. Herein, an innovative dual-chamber air-cathode microbial fuel cell was developed as biosensor for VFA monitoring. The response of the biosensor was nonlinear and increased along with the concentration of VFA mixture increase (2.8–112 mM). Meanwhile, the relationship was linear with low VFA levels (<14 mM) within 2–5 h reaction. High concentrations of bicarbonate decreased the voltage. Stirring speeded up the response and amplified the signal but reduced the saturation concentration (approximately 30 mM) and therefore narrowed the detection range. The applicability of the biosensor was further validated with the effluents from an AD reactor during a start-up period. The VFA concentrations measured by the biosensor were well correlated with the gas chromatographic measurement. The results demonstrate that this biosensor with a novel design could be used for VFA monitoring during the AD process. Based on the 16S rRNA gene sequencing, the dominant microbiomes in the biofilm were identified as Geobacter, Hydrogenophaga, Pelobacter, Chryseobacterium, Oryzomicrobium, and Dysgonomonas.