The temperature distribution inside the microchannel was first measured using a micro-Raman spectroscopy system. Polymerase chain reaction (PCR) inside the microchannel, which is very sensitive to temperature change, was chosen to validate a novel thermometry. The micro PCR chip was fabricated by silicon micromachining. The microchip consisted of a microchannel inside the chip, a Pt film heater and a Pt film sensor on the silicon surface. The O-H stretch vibration mode of water was analyzed using a micro-Raman spectroscopy system. Its spectral changes were calibrated with temperature. The intensity ratio of the hydrogen-bonded (HB) to non-hydrogen-bonded (NHB) O-H stretch vibration modes has a linear relation with temperature. The temperature of the microchannel controlled at 55 °C varied from 53.6 to 54.8 °C depending on the location of the microchannel.