Roll-to-roll (R2R) process is mass manufacturing method that can produce various products ranging from printed electronics to microfluidics. Microfluidics, used in biosensors, demands high tolerances and quality to guarantee correct functionality of sensors. In this work, optical coherence tomography device was installed at the R2R-line to measure the height and width of the hot embossed microchannel structures at speed of 1m/min. The repeating channel structure on the 10 m long sample web was measured at 1m interval. An algorithm, typically used in wavelength scanning interferometry (WSI), is utilized to recover topography at nanometer scale. The results showed that the R2R hot embossing process was very stable and predictable. If process parameters, for instance pressure at the hot embossing unit was increased, it resulted deeper channels as expected. The OCT, with the WSI algorithm, provides topography of microchannel at nanometer scale being thus an excellent tool for optimization of process parameters and for on-line quality control.