In this letter, we proposed a photoacoustic (PA) method for noninvasively detecting blood viscosity in subcutaneous microvasculature with the frequency-resolved measurement. The influence of viscosity on PA generation was investigated theoretically, and a negative correlation was shown between the viscosity and the full width at half maximum (FWHM) of the PA frequency spectrum, which can be utilized to reflect the viscosity distribution. To test the feasibility and accuracy of this method, water mixed with different concentrations of glycerol was measured. FWHM of the PA frequency spectrum was also obtained in vivo in the mouse ear to characterize the blood viscosity from different vessel bifurcations, and the metabolism-induced viscosity changes were dynamically monitored in the microvasculature. Experimental results demonstrate that this technique has future potential clinical applications for real-time monitoring the viscosity changes in subcutaneous microvasculature. Published by AIP Publishing.