Significance: Dielectrophoresis, electro-osmosis, and other electrokinetic effects are frequently used in a variety of applications but necessitate patterning of electrodes on sensor surfaces. This typically requires a cleanroom and time-consuming, expensive, and arcane lithography and etching procedures. Aim: To demonstrate the applicability of commercial laser direct writing equipment for rapid patterning of electrodes into gold layers on glass substrates, particularly with application to producing electrokinetically active plasmonic sensors. Approach: A commercial printed circuit board prototyper was used to pattern interdigitated electrode (IDE) arrays into the surface of gold-coated slides and off-the-shelf surface plasmon resonance (SPR) prisms. The electrode geometries resulting from different patterning parameters were characterized by profilometry and electron microscopy. The patterned surfaces were then employed for trapping and electro-kinetic manipulation of bacteria, and finally for sensing of bacteria by SPR imaging. Results: Fabrication of an IDE array can be completed in as little as 12 s, with longer fabrication times permitting superior geometry and minimum feature size of 15 μm. The patterned IDEs were capable of concentrating bacteria and controlling their position on the sensor surface as a function of applied frequencies. SPR was demonstrated to detect specific interactions between bacteria and immobilized antimicrobial peptides. Conclusions: Laser direct writing is demonstrated as a feasible, cleanroom-free alternative to more lengthy lithography methods, permitting very rapid fabrication and prototyping of IDEs, which are compatible with active plasmonic sensing and bacterial detection.