This work presents sensitive detection of bacterial genomic DNA using a magnetic nanoparticle-based substrate-free method. For the first time, such a method is employed for detection of a clinically relevant analyte by implementing a solid-phase-based molecular probing and amplification protocol that can be executed in 80 min. The molecular detection and amplification protocol is presented and verified on samples containing purified genomic DNA from Escherichia coli cells, showing that as few as 50 bacteria can be detected. This study moves the use of volume-amplified magnetic nanoparticles one step further toward rapid, sensitive, and selective infectious diagnostics.