Lactic acid bacteria (LAB) are recognized as probiotics worldwide. The cell itself and all kinds of metabolites are essential parts of the human life processes. The bacterial biofilm has been described as sessile bacterial communities that attached to surfaces aggregating in a hydrated polymeric matrix of their own synthesis．LAB biofilm has broad perspective for their particularities. Lactobacillus pentosus, which isolated from the traditional dairy products of Xinjiang and had a strong positive biofilm formation, was taken as the research subjects. Semi-quantitative assay was performed to examine effects of different physical and chemical factors and several food additives on biofilm formation by L. pentosus. The results showed that the optimum physical and chemical conditions for biofilm formation of L. pentosus were inoculation volume at 1∶200, incubation temperature at 37℃, culture period at 48h, glucose concentration at 2%, pH value at 5.8~6.8, and so on. The addition of Ca2+ and lactic acid obviously inhibited biofilm formation by L. pentosus completely.