Surface-based sensors that rely on diffusion for transport of target molecules to the sensor surface can lead to long and sometimes impractical detection time for low analyte concentrations. Here we describe a new method for rapid in situ SERS detection of ultralow subpicomolar concentration of the analyte molecules. The method is based upon a dynamic dielectrophoresis-enabled assembly of metal nanoparticles in the form of pearl chains with nanometer-sized gaps. We demonstrate in situ SERS measurement of benzenethiol in less than 2 min without the requirement of long incubation times. This approach is then extended to detect the biological analyte, adenine, at femtomolar concentrations in a short time from a 2 μL sample droplet.