The first demonstration of a photothermal heterodyne interferometer (PHI) combined with micro-HPLC (high-performance liquid chromatography) is reported. A semiconductor laser (375 nm) was used for excitation, and the temperature change caused by heat released from photoexcited species was detected with a He-Ne laser (632.8 nm). The temperaturedependent refractive index change of the solvent modified the optical path of the probe beam. The phase difference between two arms of the interferometer, one passing through the heated sample and another as a reference, was sensitively detected with the PHI. The nitro-polycyclic aromatic hydrocarbon and vitamin mixture separated via micro-HPLC was successfully detected with the PHI as well as a UV detector. The detection limit of the PHI for riboflavin in the absorbance units was 77 times better than that of the commercial UV detector. The detection limit of the PHI with a small flow cell (6 nL) was the same as that with a large flow cell (18 nL) for 1-nitropyrene.