In this study, we investigated the feasibility of using photoacoustic time-frequency spectral analysis (PA-TFSA) for evaluating the bone mineral density (BMD) and bone structure. Simulations and
ex vivo
experiments on bone samples with different BMDs and mean trabecular thickness (MTT) were conducted. All photoacoustic signals were processed using the wavelet transform-based PA-TFSA. The power-weighted mean frequency (PWMF) was evaluated to obtain the main frequency component at different times. The
y-intercept
,
midband-fit
, and
slope
of the linearly fitted curve of the PWMF over time were also quantified. The results show that the osteoporotic bone samples with lower BMD and thinner MTT have higher frequency components and lower acoustic frequency attenuation over time, thus higher
y-intercept
,
midband-fit
, and
slope
. The
midband-fit
and
slope
were found to be sensitive to the BMD; therefore, both parameters could be used to distinguish between osteoporotic and normal bones (
p
< 0.05).