In the present paper, the optical wavelength dependence on the photoacoustic (PA) assessment of the pulsatile blood flow was investigated by means of the experimental and theoretical approaches analyzing PA radiofrequency spectral parameters such as the spectral slope (SS) and mid-band fit (MBF). For the experimental approach, the pulsatile flow of human whole blood at 60 bpm was imaged using the VevoLAZR system (40-MHz-linear-array probe, 700-900 nm illuminations). For the theoretical approach, a Monte Carlo simulation for the light transmit into a layered tissue phantom and a Green’s function based method for the PA wave generation was implemented for illumination wavelengths of 700, 750, 800, 850 and 900 nm. The SS and MBF for the experimental results were compared to theoretical ones as a function of the illumination wavelength. The MBF increased with the optical wavelength in both theory and experiments. This was expected because the MBF is representative of the PA magnitude, and the PA signal from red blood cell (RBC) is dependent on the molar extinction coefficient of oxyhemoglobin. On the other hand, the SS decreased with the wavelength, even though the RBC size (absorber size which is related to the SS) cannot depend on the illumination wavelength. This conflicting result can be interpreted by means of the changes of the fluence pattern for different illumination wavelengths. The SS decrease with the increasing illumination wavelength should be further investigated.