The combination of electrophoretic and magnetic manipulations with electrochemical detection for a versatile microfluidic and bead-based biosensing application is demonstrated. Amperometric detection is performed in an off-channel setup by means of a voltammetric cell built at the microchannel outlet and using a gold working electrode. Superparamagnetic particles are introduced and handled inside the channel by means of an external permanent magnet in combination with the electrogenerated flow which allows reproducible loading. The specific detection of phenol as electroactive alkaline phosphatase product is used in this study as proof of concept for a sensitive protein quantification. Characterizations and optimization of different parameters have been carried out in order to achieve the best detection signal. The applicability of the device has been finally demonstrated by the detection of rabbit IgG as model protein after an immunoassay performed on magnetic particles as immobilization platform. A comparison between the electrochemical detection using the developed device and the optical standard detection revealed similar performances with, however, extremely lower amount of reagent used and shorter analysis time. The developed electrophoretic- and magnetic-based chip may open the way to several other biosensing applications with interest not only for other proteins but also for DNA analysis, cell counting, and environmental control.