Lactobacillus extracellular proteins are key molecular factors in promoting the probiotichost crosstalk and signal transduction. Their extractions and identifications could contribute to unravelling the probiotic molecular mechanisms such as adhesion to intestinal surfaces, modulating immune response and enhancing antagonism towards pathogens. A protocol based on trichloroacetic acid (TCA)acetone precipitation of proteins was presented and optimized. The method was tested on seven Lactobacillus strains after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The extracellular proteins secretion capacity of L. paracasei SW2 in different growth phases was studied and some protein bands were selected for identification by tandem MS. The extracellular proteome of L. paracasei L14 were analyzed by 2-D electrophoresis (2-DE). The results showed that the final concentration of 6% TCA was the most suitable for extracting extracellular proteins from lactobacilli. More extracellular proteins could be extracted in the stationary phase than midexponential growth phase. Three of extracellular proteins extracted from L. paracasei SW2 were successfully identified. The extracellular samples extracted from semidefined medium (SDM) could be analyzed by 2-DE. 130±10 spots, corresponding to about 46% coverage of the predicted secretome of L. paracasei, were detected in the 2-DE map of L. paracasei L14 extracellular proteome. In conclusion, this study preliminarily established the extraction method and 2-DE analysis system of extracellular proteins from lactobacilli and paved the way for a more comprehensive insight into relevant research.