The heat action spectrum of Rhodamine B by two-photon absorption has been measured by a new system of optical-probing photoacoustic spectroscopy (OPPAS) coupled with a nanosecond near-infrared OPO laser. The highly resolved spectrum was obtained in the wide spectral range from 680 nm to 900 nm. The two-photon absorption cross-section of Rhodamine B was successfully determined by comparing the signal intensity of Rhodamine B with that of the reference. The OPPAS system was able to measure a well-resolved two-photon absorption spectrum of both fluorescent and non-fluorescent molecules in the wider spectral range from visible to near-infrared region.