Abstract: The adhesion on the food surface of lactic acid bacteria (LAB) plays an important role in their colonization and biological control. The inoculated conditions of LAB Lac 9-3 (including the inoculum concentration, bacterial growth stage, soaking time, and inoculum pH) were optimized using the random-centroid optimization (RCO) method. Meanwhile, the effect of LAB Lac 9-3 on the growth and microflora of spoilage bacteria of Litopenaeus vannamei was also investigated using the high throughput sequencing technology during the storage time at 0, 8, and 14 days. The results indicated that the optimal inoculated conditions of LAB Lac 9-3 were as follows: The inoculum concentration was 10⁸ CFU/mL, the growth stage was 14.1 h (corresponding to the early stabilization period), the soaking time was 17.41 min, and the inoculum pH was 6.51. The adhesion quantity of LAB Lac 9-3 reached 2.08×10⁹ CFU/g under the optimal inoculated conditions, which obtained a 72.65% increase compared to that of the control group at the same concentration. Besides, the growth of Shewanella, Carnobacterium, Aliivibrio, and Vibrio in L. vannamei treated by LAB Lac 9-3 was significantly inhibited, indicating that improving the adhesion of LAB Lac 9-3 could increase the adhesion quantity on the surface of L. vannamei. The LAB Lac 9-3 might process the application potential in food preservation due to its regulating role in microflora and the spoilage process of aquatic products.