As rheological properties are substantially influenced by red blood cells (RBCs) and plasma, the separation of their individual contributions in blood is essential. The estimation of multiple rheological factors is a critical issue for effective early detection of diseases. In this study, three rheological properties (i.e., viscoelasticity, RBC aggregation, and blood junction pressure) are measured by analyzing the blood velocity and image intensity in a microfluidic device. Using a single syringe pump, the blood flow rate sets to a pulsatile flow pattern (Qb[t] = 1 + 0.5 sin(2πt/240) mL/h). Based on the discrete fluidic circuit model, the analytical formula of the time constant (λb) as viscoelasticity is derived and obtained at specific time intervals by analyzing the pulsatile blood velocity. To obtain RBC aggregation by reducing blood velocity substantially, an air compliance unit (ACU) is used to connect polyethylene tubing (i.d. = 250 µm, length = 150 mm) to the blood channel in parallel. The RBC aggregation index (AI) is obtained by analyzing the microscopic image intensity. The blood junction pressure (β) is obtained by integrating the blood velocity within the ACU. As a demonstration, the present method is then applied to detect either RBC-aggregated blood with different concentrations of dextran solution or hardened blood with thermally shocked RBCs. Thus, it can be concluded that the present method has the ability to consistently detect differences in diluent or RBCs in terms of three rheological properties.