The immunosensor for rapid determination of Enterobacter sakazakii in infant formula powders was constructed by employing polyamidoamine（PAMAM） and reductive grapheme as electroactive nano composites to immobilize horseradish peroxidase（HRP） labelled E. sakazakii antibody on the surface of glassy carbon electrode （GCE）. The optimum analytical parameters for determination of E. sakazakii as follows： using K3[Fe（CN）6] as probe， hydrogen peroxide concentration of base solution was 0.5 mmol/L， the pH of PBS solution was 7.0， incubation temperature was 37 ℃ and incubation time was 20 min. The relationship between the immune response currents and the concentration of Enterobacter sakazakii was studied by differential pulse voltammetry. The results showed that a linear relationship was found between the immune response current and the concentration of E. sakazakii （ΔIpc（μA） = 1.1817lgC-1.723， R2 = 0.9989）， and the limit of detection was 5.8 × 101 CFU/mL （S/N = 3）. The developed method was applied to determine E. sakazakii concentration of infant formula powders， the results were well agreed with those obtained by national standard method， and the method had the advantages of simple sample pretreatment， high sensitivity， rapid detection and low cost. Therefore， it was applicable for the rapid detection of Enterobacter sakazakii in infant formula powders.