Objective:To conjugate anti-EMMPRIN monoclonal antibody on the surface of carboxyl-SPIO nanoparticles. The efficiency of superparamagnetic iron oxide (SPIO) conjugated with anti-EMMPRIN monoclonal antibody and the size of anti-EMMPRIN-SPIO were investigated in different pH values. Methods:Anti-EMMPRIN monoclonal antibody conjugated SPIO nanoparticles were performed through a coupling strategy of EDC and sulfo-NHS. Bradford assay and SDS-PAGE analysis were used for measuring the conjugation efficiency. The size of anti-EMMPRIN-SPIO was evaluated by dynamic light scattering method. Results:The smallest diameter of 63.15 nm was obtained when pH 7.8 was used. The lower concentration of antibody in supernatant with pH6.8 and pH7.3 was 1.943 2 × 10-4 g/L and 3.511 1 × 10-4 g/L, respectively. Anti-EMMPRIN mAb was detected in lanes of pH6.8, pH7.3 and pH7.8 which revealed two bands with weight of 55 000 and 25 000. Conclusion:EDC/sulfo-NHS method is an efficient method for conjugating anti-EMMPRIN monoclonal antibody on SPIO nanoparticles. Under the amidation condition with pH value of 6.8, 7.3 and 7.8, higher conjugation efficiency and smaller size of anti-EMMPRIN-SPIO were obtained. The results showed that pH value is a very important factor which influences conjugation efficiency and the size of anti-EMMPRIN-SPIO.