Abstract: The leaves and petioles of Fraxinus mandshurica seedlings were used as research materials to induce callus. Then the growth, inoculation amount, pH and the concentration of carbon, nitrogen and phosphorus of the medium were optimized to establish a suspension cell culture system. It was found that the callus induction rate of petiole was up to 90.91% cultured on the WPM medium supplemented with 6 mg/L TDZ+5 mg/L 6−BA, while the rate for leaf was up to 87.5% cultured on the WPM supplemented with 6 mg/L TDZ. The loose callus was successfully induced on the the WPM medium supplemented with WPM+0.3 mg/L TDZ+1.5 mg/L 6−BA+0.1 mg/L NAA+0.5 mg/L IBA. The growth period of suspension cells was 18d. The fastest growth was found at 3% sugar, which increased 400% in fresh weight in a growth period. Higher concentrations of nitrogen and phosphorus were bad for the growth of the cells. A suspension cell culture system was established, which cells can be subcultured for generations.