Microfluidic mixing in combination with single-molecule spectroscopy allows the investigation of complex biomolecular processes under non-equilibrium conditions. Here we present a protocol for building, installing and operating microfluidic mixing devices optimized for this purpose. The mixer is fabricated by replica molding with polydimethylsiloxane (PDMS), which allows the production of large numbers of devices at a low cost using a single microfabricated silicon mold. The design is based on hydrodynamic focusing combined with diffusive mixing and allows single-molecule kinetics to be recorded over five orders of magnitude in time, from 1 ms to similar to 100 s. Owing to microfabricated particle filters incorporated in the inlet channels, the devices provide stable flow for many hours to days without channel blockage, which allows reliable collection of high-quality data. Modular design enables rapid exchange of samples and mixing devices, which are mounted in a specifically designed holder for use with a confocal microscopy detection system. Integrated Peltier elements provide temperature control from 4 to 37 degrees C. The protocol includes the fabrication of a silicon master, production of the microfluidic devices, instrumentation setup and data acquisition. Once a silicon master is available, devices can be produced and experiments started within similar to 1 d of preparation. We demonstrate the performance of the system with single-molecule Forster resonance energy transfer (FRET) measurements of kinetics of protein folding and conformational changes. The dead time of 1 ms, as predicted from finite element calculations, was confirmed by the measurements.