As an immunodepressant, anti-CD25 monoclonal antibody has a huge market with wide prospect and economic value. We developed a low protein serum-free medium for large-scale GS-NS0 myeloma cell culture and anti-CD25 monoclonal antibody production. Further study focused on the characteristics of GS-NS0 cell growth, glucose and amino acid metabolism, and antibody production. In the serum-free medium, the maximal viable cell density and antibody concentration reached above 3×106 cells/mL and 300 mg/L in batch culture. Compared with the commercial serum-free medium (Excell 620 + 0.2% Primatone), the maximal viable cell density doubled and the maximal antibody concentration increased 46%. Results also showed the specific growth rate decreased when the glucose concentration was lower than 6 mmol/L. And the production of lactate increased when glucose concentration was excessively high (>30 mmol/L). These results were important to provide technique and theory basis for developing optimized GS-NS0 cell culture and anti-CD25 monoclonal antibody production processes.