Integrating analytical systems in 101-103 nm spaces provides ultrasensitive analytical devices at the single cell and the single molecule levels due to the ultrasmall space, and fundamental technologies for nanofluidics are developed. A simple and ultrasensitive detection method is one of the essential technologies for nanofluidics; however, it is still challenging due to the ultrasmall volume at the attoliter to femtoliter scale. In this study, we report a new photothermal detection method of nonfluorescent molecules for a 102 nm space, photothermal optical diffraction (POD), which utilizes light absorption and heat generation by an analyte and optical diffraction by a nanochannel after heat diffusion. Concentration determination of nonfluorescent molecules in a 400 nm channel was successfully demonstrated, and a limit of detection (LOD) of 5.0 μM was achieved, corresponding to 500 molecules (0.84 zmol) in a detection volume of 230 aL. Also, detection in a 200 nm channel was successfully demonstrated without degradation of the LOD. Our method can be widely used for chemical and biological analyses in 102-103 nm nanofluidics.