Abstract: To explore the preparation and properties of seedling trays with large-size straw fibers, wheat straw was selected as raw material, and four typers of pretreatments, i.e. biological pretreatment, biological pretreatment plus MnO₂, biological pretreatment plus KOH, biological pretreatment plus MnO₂ and KOH, were applied to obtain large size straw fibers. The resulting fibers labeled as CS, CSM, CSK, and CSMK were then used to produce seedling trays through negative pressure suction filtration molding method. Characteristics of the straw fibers obtained under different pretreatment conditions and their effects on the preparation and properties of seedling trays were studied. The results showed that the average lengths of fibers of CS, CSM, CSK and CSMK were 2.20, 1.95, 1.70 and 1.55 mm, and the average widths were 96, 94, 94 and 86 µm, respectively. Mechanical properties of the seedling trays made from the obtained fibers showed that the dry and wet tensile strengths increased with fibers that pretreated with MnO₂ or/and KOH. CSMKT trays exhibited the highest mechanical strength among all samples tested, with a dry tensile strength that was 128% greater and a wet tensile strength that was approximately twice that of the trays made from CS fibers. In terms of water resistance, it was observed that the maximum water absorption rates of the various trays after 240 minutes were 629% for CST, 519% for CSMT, 524% for CSKT, and 523% for CSMKT. The results indicated that the seedling trays fabricated from straw fibers treated with MnO₂ and/or KOH exhibited superior bonding properties, resulting in enhanced structural integrity under water conditions. Solubility and swelling tests results showed that significant dissolution and swelling were observed for CST trays, indicating its relatively loose structural configuration compared to that of the more tightly bonded CSMT, CSKT, and CSMKT samples. Notably, lowest dissolution rate as well as swelling rate were observed for CSMKT, demonstrating its enhanced structural cohesion and dimensional stability. In particular, the thickness swelling rate of CSMKT was 1.26%, which was more than 50% lower than that of CST trays, highlighting its potential to maintain structural integrity under varying moisture conditions. A comparative seedling nursery test was conducted using CSMKT as experimental group and plastic seedling trays as control. It was found that the seedlings grew well in the straw fiber-based trays, with seedling strength index of cucumber, tomato, croissant, and chili pepper increased by 46.67%、61.54%、76.92% and 171.43% compared to that of the control group. Besides, the SPAD value and root vitality of the seedlings of experimental group were generally higher than that of the plastic trays by more than 10% and 38%, respectively. A cost calculation regarding material and energy consumption under pilot conditions showed that the production cost of the seedling trays prepared with large size straw fiber under different pretreatment conditions ranged from 0.49 yuan/piece to 0.51 yuan/piece, indicating good economic viability. This study showed that the large-size wheat straw fibers obtained by biological fermentation coupled with mechanical disc milling could be made into seedling tray by suction filtration method. The addition of MnO₂ or/and KOH on the basis of biological fermentation could significantly promote the deaggregation of fiber bundles, making the fiber size distribution more uniform, which was more conducive to enhancing the mechanical properties and water resistance of the seedling trays.