Botulinum neurotoxin (BoNT) poses a significant threat to public health as the most potent toxin known to humans. BoNT intoxication can lead to botulism, a life-threatening disease that may result from ingesting food contaminated with biologically active BoNT. The gold standard method for BoNT detection, the mouse bioassay, is time-consuming and raises ethical concerns regarding the use of laboratory animals. Therefore, there is a critical need for alternative methods that are rapid and require minimal training for field use. In this study, with potential in-field use in mind, we developed a microfluidic nanobiosensor that utilizes droplet-based passive pumping for easy operation and quantum dots (QDs) for sensitive detection. With droplet-based passive pumping, sensor preparation reagents and detection samples can be introduced via simple pipetting. Meanwhile, QDs offer a high quantum yield and excellent photostability, providing strong and reliable photoluminescent signals. The QDs were tethered to the surface of microfluidic channels via BoNT serotype-specific peptides, which can be cleaved due to the BoNT endopeptidase activity. In the presence of the active toxin, peptide cleavage released the QDs from the channels and the resulting reduced QD photoluminescence was measured as a detection signal. Using this biosensor, we detected the biologically active catalytic domain (light chain) of BoNT serotype A (LcA) at concentrations ranging from 1 ng/mL to 100 μg/mL, with a limit of detection (LoD) of 1 ng/mL (20 pM) in a small sample volume (1 pg LcA in 1 μL), within 2 h. This low-cost, user-friendly biosensor provides a rapid and effective method for detecting biologically active BoNT based on its endopeptidase activity without the use of laboratory animals, and it offers a scalable solution for potential BoNT screening in field or low-resource settings after further validation.