To screen suitable application modes of bio-fertilizer in rice production, this study used the late japonica rice cultivar Shanghaishida 19 as the test material and conducted field plot experiments to systematically investigate the effects of different bio-fertilizer application methods on rice yield, nitrogen accumulation and translocation, and nitrogen use efficiency(NUE). Six fertilization treatments were designed: T1, no nitrogen control; T2, conventional nitrogen application (pure N 225 kg/hm²); T3, T2 + foliar spraying of bio-fertilizer at 3.75 L/hm² at the 2-leaf-1-heart stage of seedlings; T4, T2 + foliar spraying of bio-fertilizer at 7.5 L/hm² at the 2-leaf-1-heart stage of seedlings; T5, T2 + root-dipping with bio-fertilizer at 3.75 L/hm² before transplanting; and T6, T2 + root-dipping with bio-fertilizer at 7.5 L/hm² before transplanting. The results showed that, compared with T2, co-application of bio-fertilizer significantly increased rice yield. Among these, treatment T6 achieved the highest yield, reaching 9 858.0 kg/hm², representing a significant increase of 13.74% over T2, and its grains per panicle (97.1 grains) were significantly higher than those of other treatments (P<0.05). T6 also significantly increased stem-leaf nitrogen content and nitrogen accumulation at the tillering peak stage, panicle nitrogen content at the full heading stage, panicle nitrogen accumulation at maturity, and total plant nitrogen accumulation, while effectively reducing stem-leaf nitrogen retention rate. Regarding nitrogen use efficiency, the nitrogen recovery efficiency (47.40%), nitrogen agronomic efficiency (8.34 kg/kg), and nitrogen partial factor productivity (43.81 kg/kg) of T6 were significantly higher than those of T2 by 64.01%, 171.66%, and 13.73%, respectively. In conclusion, based on conventional nitrogen application, root-dipping with bio-fertilizer at 7.5 L/hm² (diluted 5 times) before rice transplanting can effectively promote nitrogen accumulation in the early growth stage and its translocation to panicles, synergistically enhance rice yield and nitrogen use efficiency. This model is suitable as a bio-fertilizer application technology for promotion and application in green rice production.