This paper attempts to experimentally and analytically quantify the aggregation-induced changes in the photoacoustic amplitude (PAA) by simultaneously examining the effect of red blood cell (RBC) aggregate size and optical illumination wavelength. In experiments, the pulsatile flow of human whole blood at 60 bpm was imaged using the VevoLAZR system equipped with a 40-MHz-linear-array probe. The samples were illuminated every 10 nm from 700 to 900 nm. For the analytical model, the PAA from both a collection of randomly distributed RBCs of 5, 10, 15, 20, 25, and 30 cells and a single absorber as a spherical aggregate of RBCs formed by the corresponding number of RBCs. The oxygen saturation (sO2) was measured as 74% and 80% for the non-aggregated RBCs and the RBC aggregation. These values were assigned to the analytical RBC aggregates containing between 5 and 30 cells. The normalized PAA (nPAA) for the experimental results was compared to that generated by the theoretical calculations. At a given wavelength, the analytical nPAA for the collection of RBCs were identical for all numbers of RBCs, but that for the RBC aggregate increased with the number of RBCs forming the aggregate due to the increase in the effective photoacoustic absorber size. The experimental as well as analytical nPAA for both RBC aggregation and non-aggregation increased with the wavelength at a given absorber size. This was due to the fact that the PAA is mainly determined by the optical absorption coefficient (μa) which increases due to the relationship between εHbO and wavelength. In addition, the difference of PAA between RBC aggregation and nonaggregation also increased with the wavelength due to the increase in the μa induced by the hypothesized enhanced sO2 resulting from the increased size of RBC aggregates. These results can be used as a means of estimating the oxygen loading and unloading during blood flow. This investigation elucidates the quantitative relationship between the RBC aggregate size and the optical illumination wavelength for probing the physiology of flowing blood.