In this study, we established a versatile and simple magnetic-assisted microfluidic method for fast bacterial detection. Quantum dots (QDs) were loaded onto magnetic beads (MBs) to construct performance enhanced on-chip capture of bacteria. Escherichia coli (E. coli), as a model bacterium was studied. CdSe QDs were deposited onto the surface of Fe3O4 MBs through layer-by-layer self-assembly to enhance the loading of antibodies (Abs). MBs functionalized with anti-E. coli antibody molecules in a micropillar-based microfluidic chip were utilized to capture E. coli, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for characterization of captured bacteria. This method was found capable of specifically isolating E. coli within the range of 1.0 to 1.0 × 109 CFU/mL, having a detection limit (LOD) of 10 CFU/mL. The average similarity score among mass spectra for the bacterial capture obtained in independent experiments is calculated as 0.97 ± 0.01 (n = 3), which shows this work's excellent reproducibility for bacterial capture. Bacterial growth on ready-to-eat (RTE) foods during its time of storage was successfully monitored. The present protocol has promising potential for microbial control and pathogen detection in the food industry.