Abstract: In order to explore the optimized culture condition of mixed- species biofilm on coconut, the culture of mixed- species biofilm on coconut formed by Lactobacillus bulgaricus and Streptococcus thermophilus was optimized by single factor and orthogonal test. Four factors ( time, temperature, solid- liquid ratio, inoculation amount) were chosen.Results showed the effect of these factors on the viable count of mixed- species biofilm on coconut: temperature > time > solid- liquid ratio > inoculation amount. The optimized culture condition of mixed-species biofilm on coconut was: time 2.5d, temperature 37℃, solid-liquid ratio 1∶30 ( g /mL) , inoculation amount 2%.Under such condition, the viable count of mixed- species biofilm on coconut reached the maximum of 5.79 ×108CFU /cm2.