This paper describes the first immunosensing system reported for the detection of bacteria combining immunomagnetic capture and amperometric detection in a one-step sandwich format, and in a microfluidic environment. Detection is based on the electrochemical monitoring of the activity of horseradish peroxidase (HRP), an enzyme label, through its catalysis of hydrogen peroxide (H2O2) in the presence of the mediator hydroquinone (HQ). The enzymatic reaction takes place in an incubation micro-chamber where the magnetic particles (MPs) are confined, upstream from the working electrode. The enzyme product is then pumped along a microchannel, where it is amperometrically detected by a set of microelectrodes. This design avoids direct contact of the biocomponents with the electrode, which lowers the risk of electrode fouling. The whole assay can be completed in 1h. The experiments performed with Escherichia coli evidenced a linear response for concentrations ranging 102–108cellml−1, with a limit of detection of 55cellsml−1 in PBS, without pre-enrichment steps. Furthermore, 100cellsml−1 could be detected in milk, and with negligible interference by non-target bacteria such as Pseudomonas.