The Grüneisen parameter, a constitutive parameter in photoacoustics, is usually measured from isobaric thermal expansion, which may not be valid for a biological medium due to its heterogeneity. Here, we directly measured the Grüneisen parameter by applying photoacoustic spectroscopy. Laser pulses at wavelengths between 460 and 1800 nm were delivered to tissue samples, and photoacoustic signals were detected by flat water-immersion ultrasonic transducers. Least-squares fitting photoacoustic spectra to molar optical absorption spectra showed that the Grüneisen parameter was 0.81 ± 0.05 (mean ± SD) for porcine subcutaneous fat tissue and 0.69 ± 0.02 for porcine lipid at room temperature (22°C). The Grüneisen parameter of a red blood cell suspension was linearly related to hemoglobin concentration, and the parameter of bovine serum was 9% greater than that of water at room temperature.