This paper reports the application of temperature gradient focusing (TGF) in a PDMS/glass hybrid microfluidic chip. With TGF, by the combination of a temperature gradient along a microchannel, an applied electric field, and a buffer with a temperature-dependent ionic strength, analytes are focused by balancing their electrophoretic velocities against the bulk velocity of the buffer containing the analytes. In this work, Oregon Green 488 carboxylic acid was concentrated approximately 30 times as high as the initial concentration in 45 s at moderate electric strength of 70 V/cm and a temperature gradient of 55 degrees C across the PDMS/glass hybrid microfluidic chip with a 1 cm long capillary.