To explore the effects of bio-organic fertilizer on millet [Setaria italica (L.) Beauv] cultivation and soil fertility improvement in the Mu Us sandy land，six treatments were established:no fertilization (CK)，100% chemical fertilizer (T1)，20% bio-organic fertilizer + 80% chemical fertilizer (T2)，20% bio-organic fertilizer substrate + 80% chemical fertilizer (T3)，100% bio-organic fertilizer (T4)，and 20% organic fertilizer + 80% chemical fertilizer (T5).The study focused on millet agronomic traits，yield and its components，soil nutrients，and soil microbial community characteristics.The results showed that fertilization treatments had positive effects on millet agronomic traits，yield，and its components.Among them，T2 had the best growth and yield-promoting effects，with a yield of 4197 kg/hm²，increasing by 71% and 32%，compared to CK and T1，respectively.The yield increase effect of T4 was not significant，but it showed the best improvement in soil pH，organic matter，and nutrient content.The soil organic matter content in T2 was second only to T4，while other nutrient indices were slightly lower than those in T4.The carbon source metabolic activity of the soil microbial community showed a trend of T4>T2> T3>T1>T5>CK，with amino acids being the preferentially utilized substrates.However，the application of bio-organic fertilizer promoted the utilization of sugars and carboxylic acids.The application of bio-organic fertilizer also enhanced the diversity of soil microbial community，as indicated by the highest Shannon and Simpson indices in T2 and the highest McIntosh index in T4.Overall，the combined application of bio-organic fertilizer and chemical fertilizer was an effective method for balancing soil fertility improvement and millet yield in the Mu Us sandy land under the conditions of this study.These results could provide scientific reference for effective crop cultivation and soil improvement in sandy areas.