A selection and optimization procedure for the synbiotic combination of probiotic and prebiotics was established to optimize its cholesterol removal in vitro. In light of fermentability, prebiotics utilization by probiotics was highly variable and interspecies differences existed. Based on the results of fermentability, L. plantarum LS12, Ls31, LP529 and L. ruminis La3 could be the better candidates for symbiotic research. The bile tolerance of all the tested strains could be improved by the strain-specific prebiotics comparing to the control carbon source (glucose). The strain LS12 was finally selected to form the symbiotic according to its better ability to ferment prebiotics and bile tolerance, while the five prebiotics (FOS, stachyose, GOS, IMO and mannitol) were selected to make their synbiotic combination because of their better enhancement of bile tolerance and growth support to LS12. The synbiotic combination for cholesterol removal was optimized by use of response surface methodology. The first-order model showed that the selected prebiotics mannitol and GOS were significant factors. Then through the second-order polynomial regression model, the optimum conditions of the two factors for cholesterol removal by the synbiotic were suggested.