To improve the mechanized transplanting efficiency for large-scale broccoli production in Ningxia, this study aims to identify key morphological traits of seedlings suitable for mechanized transplanting. A Box–Behnken design was used to set three experimental factors, broccoli variety, seedling age, and plug tray specification, to evaluate their effects on seedling plant type (plant height, stem diameter, canopy diameter, stem inclination angle, and plant type cone angle) and root system characteristics (substrate loss rate). The results showed that plug tray specification was the primary factor affecting substrate loss rate, followed by variety and seedling age. Seedling age was the dominant factor affecting plant height, stem diameter, and canopy diameter, while plug tray specification primarily influenced stem inclination angle. Optimization via response surface methodology (RSM) indicated that the best transplanting performance was achieved with the “Hannai Youxiu” variety (excellent cold tolerance), 30-day-old seedlings, and 72-cell or 98-cell plug trays. Field validation confirmed that under these optimal parameters the mechanized transplanting feeding rate reached 100%, the seedling missing rate was 2.5%, and the transplanting qualification rate was 97.5%, with all RMSE values being less than 7.5%. These findings provide a scientific basis for the mechanized transplanting of broccoli in Ningxia, recommending the “Hannai Youxiu” variety and 98-cell plug trays with 30-day-old seedlings to enhance transplanting quality and production efficiency.