Osteoporosis is a progressive bone disease that is characterized by a decrease in bone mass and the deterioration in bone microarchitecture. This study investigates the feasibility of characterizing bone microstructure by analyzing the frequency spectrum of the photoacoustic (PA) signal from the bone. Modeling and numerical simulation of PA signal were performed on trabecular bone simulations and CT scans with different trabecular thicknesses. The resulting quasi-linear photoacoustic spectra were fittted by linear regression, from which the spectral parameter slope was quantified. The simulation based on two different models both demonstrate that bone specimens with thinner trabecular thicknesses have higher slope. Experiment on osteoporotic rat femoral heads with different mineral content was conducted. The finding from the experiment was in good agreement with the simulation, demonstrating that the frequency-domain analysis of PA signals can provide an objective assessment of bone microstructure and deterioration. Considering that PA measurement is non-ionizing, non-invasive, and has sufficient penetration in both calcified and non-calcified tissues, this new bone evaluation method based on photoacoustic spectral analysis holds potential for clinical management of osteoporosis and other bone diseases.