Considering the importance of determining the levels of hemoglobin (Hb) as a vital protein in red blood cells, in this work a highly sensitive electrochemical sensor was developed based on a gold electrode (AuE) modified with Ag metal-organic framework mesoporous carbon (Ag-MOF@MC) and molecularly imprinted polymers (MIPs). To that end, the MIP layer was formed on the Ag-MOF@MC by implanting Hb as the pattern molecule during the polymerization. The modified electrode was designed using electrochemical approaches including differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). Using a response level experimental design method, the most important parameters affecting the reaction of the sensing system including pH, incubation time, and scanning rate were optimized. Following the same route, the Hb concentration, pH, temperature, and elution times were optimized to prepare the imprinted polymer layer on the Ag-MOF@MC surface. By exploiting DPV techniques based on the optimal parameters, the electrochemical response of the AuE/Ag-MOF@MC-MIPs for Hb determination was recorded in a wide linear dynamic range (LDR) of 0.2 pM to 1000 nM, with a limit of detection (LOD) of 0.09 pM. Moreover, the Ag-MOF@MC-MIP sensing system showed good stability, high selectivity, and acceptable reproducibility for Hb determination. The sensing system was successfully applied for Hb determination in real blood samples, and the results were compared with those of the standard methods for Hb determination. Acceptable recovery (99.0%) and RDS% (4.6%) confirmed the applicability and reliability of the designed Hb sensing system.