A simple and sensitive electrochemical biosensor for acrylamide (AA) detection based on hemoglobin (Hb) entrapped in ionic liquid-carbon paste was constructed. The Hb-carbon ionic liquid paste electrode (Hb/CILPE) was studied and characterized by scanning electron microscopy (SEM) and electrochemical method. The surface passivation of the Hb/CILPE could be achieved by the addition of acrylamide molecule, which caused the lower current response value owing to the adduct formed by acrylamide and Hb as a result of the reaction between the α-NH2 group and the N-terminal valine of Hb. The experimental conditions such as pH value of supporting electrolyte, pulse increment and concentrated time of AA was examined. Under the optimum conditions, the differential pulse voltammetry (DPV) responses of Hb/CILPE toward AA was evaluated in a good linear range from 1.0 × 10−11 to 1.0 × 10−5 M, with a detection limit of 5.0 × 10−12 M at S/N = 3. This biosensor can be successfully applied to the determination of AA in a real sample without complex pretreatment. Compared with other electrochemical methods for detecting AA, the prepared electrochemical biosensor exhibited advantages of simple preparation, easy surface renewal, better reproducibility and satisfactory stability.