To accurately simulate the interactions between the plug seedlings and the planter during the planting process and explore the damage mechanism of the plug seedling substrate block, the bonding parameters of plug seedlings substrate block were calibrated by the discrete element method. The physical puncture test showed that the average of maximum force of the cone indenter on substrate block measured by texture analyzer was 4.633 N. A Hertz-Mindlin with the bonding model was established for the substrate block in EDEM software. A virtual calibration experiment was designed with the puncture force of substrate block as the evaluation index. The two-level factorial test and the steepest climbing test were used to screen out the significant parameters and the optimal interval. Then, the Box-Behnken test and the optimization solution were used to obtain the optimal bonding parameter combination of the substrate block particles. Finally, the optimal parameter combination was simulated and verified. The relative error of the maximum puncture force between the simulated value and the measured value was 1.88 %, which indicated that the bonding parameters of the substrate block obtained by calibration were accurate and reliable.